Clostridial toxin activity assays

ABSTRACT

Compositions useful for detecting Clostridial toxin activity comprising a cell that contains an exogenous Clostridial toxin substrate which comprises a fluorescent member, a membrane targeting domain and a Clostridial toxin recognition sequence comprising a cleavage site, where the cleavage site intervenes between the fluorescent member and the membrane targeting domain; and methods useful for determining Clostridial toxin activity using such Clostridial toxin substrates.

This is a national stage application under 35 U.S.C. §371 of PCT patentapplication PCT/US2006/012825, filed on Apr. 4, 2006 which claims thebenefit of priority pursuant to 35 U.S.C. §119(e) to United Statesprovisional patent application Ser. No. 60/668,909 filed on Apr. 5,2005, each of which is hereby incorporated by reference in its entirety.

All of the publications cited in this application are herebyincorporated by reference herein in their entirety. All GeneBanksequence listings cited this application, as identified by their GenBankaccession numbers, are available from the National Center forBiotechnological Information and are all hereby incorporated byreference in their entirety.

The myorelaxant properties of Clostridial toxins (CoNTs) are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). For example, CoNTs therapies areproposed for treating dystonia, see e.g., Kei Roger Aoki, et al., Methodfor treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No.6,319,505 (Nov. 20, 2001); pain, see e.g., Kei Roger Aoki, et al.,Method for Treating Pain by Peripheral Administration of a Neurotoxin,U.S. Pat. No. 6,464,986 (Oct. 15, 2002); muscle injuries, see e.g.,Gregory F. Brooks, Methods for Treating Muscle Injuries, U.S. Pat. No.6,423,319 (Jul. 23, 2002); cardiovascular diseases, see e.g., Gregory F.Brooks, Methods for Treating Cardiovascular Diseases with BotulinumToxins, U.S. Patent Publication No. 2003/0185860 (Oct. 2, 2003);neuropsychiatric disorders, see e.g., Steven Donovan, TherapeuticTreatments for Neuropsychiatric Disorders, U.S. Patent Publication No.2003/0211121 (Nov. 13, 2003); lower back pain, see e.g., Kei Roger Aoki,et al., Botulinum Toxin Therapy for Lower Back Pain, U.S. PatentPublication No. 2004/0037852 (Feb. 26, 2004); as well as otherneuromuscular disorders, see e.g., Kei Roger Aoki, et al., MultipleBotulinum Toxins for Treating Neuromuscular Disorders and Conditions,U.S. Patent Publication No. 2001/0021695 (Sep. 13, 2001); Kei RogerAoki, et al., Treatment of Neuromuscular Disorders and Conditions withDifferent Botulinum, U.S. Patent Publication No. 2002/0010138 (Jan. 24,2002); Kei Roger Aoki, et al., Use of Botulinum Toxins for TreatingVarious Disorders and Conditions and Associated Pain, U.S. PatentPublication No. 2004/0013692 (Jan. 22, 2004) all of which are herebyincorporated by reference. Additional proposed uses of CoNTs asbiopharmaceutical neuromodulators has expanded to cover a wide varietyof treatments targeting certain disorders that lack a neuromuscularbasis. For example, the effects on the autonomic nervous system hasallowed the development of a Botulinum toxin serotype A (BoNT/A) therapyfor treating axillary hyperhydrosis or sweating, and reports indicateBoNT/A may be an effective treatment for myofascial pain and tension,stroke, traumatic brain injury, cerebral palsy, gastrointestinalmotility disorders, urinary incontinence cancer and migraine headaches.Lastly, cosmetic and other therapeutic applications are widely known. Infact, the expected use of CoNTs in both therapeutic and cosmetictreatments of humans is anticipated to expand to an ever widening rangeof diseases and aliments that can benefit from the myorelaxantproperties of these toxins.

The growing clinical and therapeutic use of Clostridial toxinsnecessitates the pharmaceutical industry to use accurate assays forClostridial toxin activity in order to, for example, ensure accuratepharmaceutical formulations and monitor established quality controlstandards. In addition, given the potential danger associated with smallquantities of Clostridial toxins in foodstuffs, the food industryrequires Clostridial toxin assays, for example, to validate new foodpackaging methods and to ensure food safety. The present inventionprovides novel Clostridial toxin assays for determining the presence oractivity of a Clostridial toxin useful for various industries, such as,e.g. the pharmaceutical and food industries, and provides relatedadvantages as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of the intoxicationmechanism for tetanus and botulinum toxin activity in central andperipheral neuron. This intoxication process can be described ascomprising four steps: 1) receptor binding, where Clostridial toxinbinds to a Clostridial receptor system initiates the intoxicationprocess; 2) complex internalization, where after toxin binding, avesicle containing a toxin/receptor system complex is endocytosised intothe cell; 3) light chain translocation, where multiple events arethought to occur, including changes in the internal pH of the vesicle,formation of a channel pore comprising the H_(N) domain of Clostridialtoxin heavy chain, separation of the Clostridial toxin light chain fromthe heavy chain, enzymatic activation of the light chain; and release ofthe activated light chain and 4) enzymatic target modification, wherethe activated light chain of Clostridial toxin proteolytically cleavesits target SNARE substrates, such as, e.g., SNAP-25, VAMP or Syntaxin.

FIG. 2 shows a schematic of SNARE proteins. FIG. 2 a shows the generaldomain organization of SNAP-25, VAMP and Syntaxin depicting approximatelocations of the a-helical resions (white boxs), SNARE motifs (Hatchedboxes with S1, S2, S3, S4, V1, V2, X1 or X2 designations) and themembrane anchoring domains (white boxes designated MA). FIG. 2 b shoesthe helical organization of a SNARE motif.

FIG. 3 shows a schematic of the subcellular localization and cleavagesites of SNAP-25, VAMP and Syntaxin. VAMP is localized to synapticvesicle membrane, whereas SNAP-25 and Syntaxin are localized to theplasma membrane. BoNT/A and BoNT/E cleave SNAP-25 close to thecarboxy-terminus, releasing nine or 26 residues, respectively. BoNT/B,BoNT/D, BoNT/F, BoNT/G and TeNT act on the conserved central portion ofVAMP (white box) and release the amino-terminal cytosolic half of VAMPinto the cytosol. BoNT/C1 cleaves SNAP-25 close to the carboxy-terminusas well as cleaving Syntaxin at a single site near the cytosolicmembrane surface. The action of BoNT/C1 results in release of a largeportion of the cytosolic domain of Syntaxin, while only a small portionof SNAP-25 is released by selective proteolysis of BoNT/C1.

FIG. 4 shows PC12 cells transfected with a plasmid encoding a greenfluorescent protein alone (GFP, transfected with a plasmid encoding aClostridial toxin substrate alone (GFP-SNAP25₂₀₆), or co-transfected aplasmid encoding a Clostridial toxin substrate alone (GFP-SNAP25₂₀₆) anda plasmid encoding the light chain of BoNT/A (BoNT/A-LC). Cellsexpressing green fluorescent protein alone (GFP) had fluorescencedispersed throughout the cell including the nuclei. Confocal pictureswere taken with the plane in the middle of the cell. Cells expressingthe Clostridial toxin substrate alone (GFP-SNAP25₂₀₆) demonstratedfluorescence in the plasma membrane of the cell body and neurites. Cellsco-expressing the Clostridial toxin substrate and the BoNT/A light chain(GFP-SNAP25₂₀₆, BoNT/A-LC) exhibit a loss of plasma membranelocalization of the GFP fluorescence. The GFP fluorescence insteadaccumulates in some areas of the cytoplasm.

FIG. 5 shows Western blot analysis identifying cells with high affinityuptake for a Clostridial toxin. FIG. 5 a shows a Western blot analysisused to identify cells capable of BoNT/A uptake. The blot shows fivecell lines treated with 1 nM of Pure BoNT/A overnight, with equalamounts of protein loaded per lane and probed with an antibody thatdetects the BoNT/A SNAP-25₁₉₇ cleavage product. FIG. 5 b shows Westernblot analysis used to evaluate the time necessary for BoNT/A uptake. Theblots show either Neuro-2A cells or SH-SY5Y cells treated with 1 nM ofPure BoNT/A for various lengths of time, with equal amounts of proteinloaded per lane and probed with an antibody that detects the BoNT/ASNAP-25₁₉₇ cleavage product. FIG. 5 c shows a Western blot analysis usedto evaluate the concentration range necessary of BoNT/A uptake. Theblots show Neuro-2A cells treated with a range of Pure BoNT/Aconcentrations overnight, with equal amounts of protein loaded per laneand probed with an antibody that detects the BoNT/A SNAP-25₁₉₇ cleavageproduct.

FIG. 6 shows Western blot analysis identifying cells with high affinityuptake for a Clostridial toxin. FIG. 6 a shows a Western blot analysisused to identify cells capable of BoNT/E uptake. The top blot showNeuro-2A cells and SH-SY5Y cells treated with either 10 nM or 100 nM ofBoNT/E di-chain overnight, with equal amounts of protein loaded per laneand probed with an antibody (SMI-81; Sternberger Monoclonals,Lutherville, Md.) that detects the uncleaved SNAP-25₂₀₆ substrate andthe BoNT/E SNAP-25₁₈₀ cleavage product. The bottom blot show variouscells treated with 20 nM of BoNT/E di-chain, with equal amounts ofprotein loaded per lane and probed with an antibody for the uncleavedSNAP-25₂₀₆ substrate and the BoNT/E SNAP-25₁₈₀ cleavage product. FIG. 6b shows Western blot analysis used to determine a time course for BoNT/Euptake. The blots show SH-SY5Y cells treated with either 5 nM or 20 nMof BoNT/E di-chain for either 4 hours or 8 hours, with equal amounts ofprotein loaded per lane and probed with an antibody (SMI-81; SternbergerMonoclonals, Lutherville, Md.) that detects the uncleaved SNAP-25₂₀₆substrate and the BoNT/E SNAP-25₁₈₀ cleavage product. FIG. 6 c shows aWestern blot analysis used to evaluate the concentration range necessaryof BoNT/E uptake. The blots show SK-N-DZ cells treated with a range ofBoNT/E di-chain concentrations for approximately 6 hours, with equalamounts of protein loaded per lane and probed with an antibody (SMI-81;Sternberger Monoclonals, Lutherville, Md.) that detects the uncleavedSNAP-25₂₀₆ substrate and the BoNT/E SNAP-25₁₈₀ cleavage product.

FIG. 7 shows Western blot analysis evaluating the effects of treatmentsused to increase uptake of a Costridial toxin. FIG. 7 a shows a Westernblot analysis evaluating the effects of ganglioside treatment on theuptake of BoNT/A. The blot shows Neuro-2A cells treated without or with25 μg/mL of GT1b (− or +) and exposed overnight to three differentconcentrations of BoNT/A (12.5 pM, 25 pM or 50 pM), with equal amountsof protein loaded per lane and probed with an antibody that detects theBoNT/A SNAP-25₁₉₇ cleavage product. FIG. 7 b shows Western blot analysisevaluating the effects of cell differentiation on the uptake of BoNT/A.The blots show either Neuro-2A cells or SH-SY5Y cells treated 2 nM ofPure BoNT/A overnight that where either grown in serum-free media orwith various differentiation reagents (Ionomycin, db-cAMP, Retinoicacid, Neuraminidase or N2), with equal amounts of protein loaded perlane and probed with an antibody (SMI-81; Sternberger Monoclonals,Lutherville, Md.) that detects the uncleaved SNAP-25₂₀₆ substrate andthe BoNT/A SNAP-25₁₉₇ cleavage product.

FIG. 8 shows Western blot analysis evaluating the effects of treatmentsused to increase uptake of a Clostridial toxin. FIG. 8 a shows a Westernblot analysis evaluating the effects of ganglioside treatment on theuptake of BoNT/E. The blot shows Neuro-2A cells treated with either 25μg/mL of GT1b, GQ1b, GD1a, GD1b or GD3 and exposed for approximately 5hours to 14 nM of BoNT/E di-chain, with equal amounts of protein loadedper lane and probed with an antibody (SMI-81; Sternberger Monoclonals,Lutherville, MD) that detects the uncleaved SNAP-25₂₀₆ substrate and theBoNT/E SNAP-25₁₈₀ cleavage product. FIG. 8 b shows Western blot analysisevaluating the effects of cell differentiation on the uptake of BoNT/E.The blots show either N1E-115 cells, SH-SY5Y cells, SK-N-DZ cells orNG108-15 cells treated with either 0 nM, 2 nM or 20 nM of BoNT/Edi-chain for approximately 6 hours that where grown in serum-free media,with equal amounts of protein loaded per lane and probed with anantibody (SMI-81; Sternberger Monoclonals, Lutherville, MD) that detectsthe uncleaved SNAP-25₂₀₆ substrate and the BoNT/E SNAP-25₁₈₀ cleavageproduct.

FIG. 9 shows a schematic of a fluorescent-based Clostridial toxinactivity assay which relies on cell lines containing a Clostridial toxinsubstrate localized to the cell membrane. FIG. 9 a shows an assayscenario where the uncleaved Clostridial toxin substrate, comprising afluorescent member (FM), a membrane targeting domain (MTD) and aClostridial toxin recognition sequence comprising a Clostridial toxincleavage site (RS), is detected. Upon excitation, the fluorescent memberemits fluorescent light at a characteristic wavelength that is localizedto the membrane. FIG. 9 b shows an assay scenario where the cleavedClostridial toxin substrate is detected. Upon excitation, thefluorescent member emits fluorescent light at a characteristicwavelength. However, because the cleavage product containing thefluorescent member is released into the cytoplasm, detection offluorescence is in a different subcellular localization. Thus, adecrease in fluorescent member emissions in the membrane, or an increasein fluorescent member emissions in the cytoplasm is indicative of thepresence of Clostridial toxin activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel assays for determining the presenceor absence of an active Clostridial toxin in a sample and fordetermining the activity of a Clostridial toxin, including botulinumtoxins of all serotypes and tetanus toxin. The novel assays of theinvention rely on the cellular localization of the uncleaved Clostridialtoxin substrate, the cellular localization of the cleaved Clostridialtoxin substrate, or the cellular localization of both the uncleaved andcleaved Clostridial toxin substrate. The present invention furtherprovides novel compositions, including cells and cell lines containingClostridial toxin substrates, useful for the assays disclosed in thespecification. The novel cells and assays of the invention reduce theneed for animal toxicity studies, yet serve to analyze multiple toxinfunctions, namely, binding and cellular uptake of toxin, translocationinto the cell cytosol, and protease activity. As discussed furtherbelow, the novel cells and methods of the invention can be used toanalyze crude and bulk samples as well as highly purified dichain toxinsand formulated toxin products and further are amenable to automated highthroughput assay formats.

The assays disclosed in the present specification use cells which arecapable of efficient Clostridial toxin uptake and which include amembrane localized Clostridial toxin substrate containing a fluorescentmarker. As an example, a cell useful in the invention can express aSNAP25₂₀₆-enhanced green fluorescent protein (EGFP) fusion protein(absorbance 484 nM, emission 510 nM), which localizes to the plasmamembrane (FIG. 9). Upon BoNT/A treatment of this cell, cleavage of themembrane localized SNAP25₂₀₆-EGFP substrate occurs, releasing the EGFPcontaining fragment into the cytoplasm. Upon excitation of the treatedcell with a 484 nM laser, the EGFP is excited and emits light at 510 nM.However, because a portion of the EGFP is now cytoplasmic, adistribution change between the uncleaved, membrane localizedSNAP25₂₀₆-EGFP toxin substrate and the cleaved, cytoplasmic localizedEGFP fragment can be observed in BoNT/A treated cells.

Aspects of the present invention provide for an exogenous Clostridialtoxin substrate capable of being localized to the plasma membrane of acell. These substrates comprise a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain.

Other aspects of the present invention provide compositions comprising acell containing an exogenous Clostridial toxin substrate capable ofbeing localized to the plasma membrane of the cell wherein the cell iscapable of Clostridial toxin intoxication, and wherein the exogenousClostridial toxin substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain. The exogenousClostridial toxin substrate can be transiently or stably contained inthe cell.

Other aspects of the present invention provide compositions comprising acell population, said cell population comprising cells that contain anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of the cells wherein the cells are capable ofClostridial toxin intoxication, and wherein the exogenous Clostridialtoxin substrate comprises a fluorescent member, a membrane targetingdomain and a Clostridial toxin recognition sequence comprising acleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain and whereingreater than 50% of the cell population comprise the cells containingthe exogenous Clostridial toxin substrate.

Other aspects of the present invention provide compositions comprising acell population, said cell population comprising cells that transientlycontain an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of the cells wherein the cells arecapable of Clostridial toxin intoxication, and wherein the exogenousClostridial toxin substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain and whereingreater than 50% of the cell population comprise the cells containingthe exogenous Clostridial toxin substrate.

Other aspects of the present invention provide compositions comprising acell population, the cell population comprising cells that stablycontain an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of the cells wherein the cells arecapable of Clostridial toxin intoxication, and wherein the exogenousClostridial toxin substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain.

Other aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that contain anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of the cells wherein the cell are capable of Clostridialtoxin intoxication, wherein the exogenous Clostridial toxin substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between the fluorescent member and themembrane localization domain and wherein greater than 50% of the cellpopulation comprises the cells containing the exogenous Clostridialtoxin substrate; exciting the fluorescent member; and determining thefluorescence of the contacted cell population relative to a control cellpopulation, where a difference in fluorescence of the contacted cellpopulation as compared to the control cell population is indicative ofClostridial toxin activity.

Other aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that transientlycontains an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of the cells wherein the cells arecapable of Clostridial toxin intoxication, wherein the exogenousClostridial toxin substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between thefluorescent member and the membrane localization domain and whereingreater than 50% of the cell population comprises the cells containingthe exogenous Clostridial toxin substrate; exciting the fluorescentmember; and determining the fluorescence of the contacted cellpopulation relative to a control cell population, where a difference influorescence of the contacted cell population as compared to the controlcell population is indicative of Clostridial toxin activity.

Other aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that stably contain anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of the cells wherein the cells are capable ofClostridial toxin intoxication, wherein the exogenous Clostridial toxinsubstrate comprises a fluorescent member, a membrane targeting domainand a Clostridial toxin recognition sequence comprising a cleavage site,where the cleavage site intervenes between the fluorescent member andthe membrane localization domain and wherein greater than 50% of thecell population comprises the cells containing the exogenous Clostridialtoxin substrate; exciting the fluorescent member; and determining thefluorescence of the contacted cell population relative to a control cellpopulation, where a difference in fluorescence of the contacted cellpopulation as compared to the control cell population is indicative ofClostridial toxin activity.

Bacteria of the genus Clostridia are strictly anaerobic to aero-tolerantspore-forming bacilli found in soil, freshwater and saltwater sediments,household dust, the surface of foods, feces as well as in the normalintestinal flora of humans and animals. While the majority of isolatesare gram-positive, a few gram-negative species exist. Members of thisgenus produce sophisticated exotoxins that are among the most potenttoxins known in the world. Exposure to these toxins during the course ofClostridia infection is the primary cause underlying diseasepathogenesis. Clostridia are a major threat to human and animal health,being responsible for many diseases including botulism, tetanus, gasgangrene, pseudomembranous colitis and food poisoning. For example,Clostridium argentinense, C. bifermentans, C. histolyticum, C. novyi, C.septicum, C. sporogenes and C. tertium are etiological agents for gasgangrene. C. perfringens is responsible for foodborne illness, enteritisnecroticans where as C. difficile is responsible for pseudomembranousenterocolitis. Both C. baratii and C. butyricum are causative agents fora form of foodborne, intestinal and wound botulism. Interestingly, onlya few species of these bacteria are pathogenic for humans, most aresaprophytic. Thus, in most cases, Clostridia are opportunistic pathogensthat infect a host whose health is compromised.

Of all Clostridia, Clostridium botulinum and Clostridium tetani producethe most potent biological toxins known and are the causative agents ofthe neuroparalytic syndromes botulism and tetanus. Sevenantigenically-distinct types of Botulinum toxins (BoNTs) have beenidentified by investigating botulism outbreaks in man (BoNT/A, /B, /Eand /F), animals (BoNT/C1 and /D), or isolated from soil (BoNT/G). BoNTspossess approximately 35% amino acid identity with each other and sharethe same functional domain organization and overall structuralarchitecture. The amino acid sequences of eight Clostridial toxinserotypes have been derived from the corresponding genes (Niemann,“Molecular Biology of Clostridial Neurotoxins” in Sourcebook ofBacterial Protein Toxins Alouf and Freer (Eds.) pp. 303-348 London:Academic Press 1991). It is recognized by those of skill in the art thatwithin each type of Clostridial toxin there can be various strainsdiffering somewhat in their amino acid sequence, and also in the nucleicacids encoding these proteins. While all seven BoNT serotypes havesimilar structure and pharmacological properties, each also displaysheterogeneous bacteriological characteristics. In contrast, tetanustoxin (TeNT) is produced by a uniform group of C. tetani. Two otherspecies of clostridia, C. baratii and C. butyricum, also produce toxinssimilar to BoNT/F and BoNT/E, respectively.

Clostridia toxins (CoNTs) are each translated as a single chainpolypeptide of approximately 150 kDa that is subsequently cleaved byproteolytic scission within a disulphide loop by bacterial or tissueproteases. This posttranslational processing yields a di-chain moleculecomprising an approximately 50 kDa light chain (LC) and an approximately100 kDa heavy chain (HC) held together by a single disulphide bond andnoncovalent interactions. Each mature di-chain molecule comprises threefunctionally distinct domains: 1) an enzymatic domain located in the LCthat includes a metalloprotease region containing a zinc-dependentendopeptidase activity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the toxin from intracellular vesicles into the cytoplasm ofthe target cell; and 3) a binding domain found within thecarboxy-terminal half of the HC (H_(C)) that determines the bindingactivity and binding specificity of the toxin to the receptor complexlocated at the surface of the target cell.

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby CoNTs enter a neuron and inhibitneurotransmitter release is similar, regardless of type. Although theapplicants have no wish to be limited by the following description, theintoxication mechanism can be described as comprising four steps: 1)receptor binding, 2) complex internalization, 3) light chaintranslocation, and 4) enzymatic target modification (see FIG. 1). Theprocess is initiated when the H_(C) domain of a CoNT binds toCoNT-specific receptor complex located on the plasma membrane surface ofa target cell. The binding specificity of a receptor complex is thoughtto be achieved, in part, by specific combinations of gangliosides andprotein receptors that appear to distinctly comprise each Clostridialtoxin receptor complex. Once bound, the CoNT/receptor complexes areinternalized by endocytosis and the internalized vesicles are sorted tospecific intracellular routes. The translocation step appears to betriggered by the acidification of the vesicle compartment. This processseems to initiate two important pH-dependent structural rearrangementsthat increase hydrophobicity and promote enzymatic activation of thetoxin. Once activated, light chain endopeptidase of the toxin isreleased from the intracellular vesicle into the cytosol where itspecifically targets one of three known core components of theneurotransmitter release apparatus. There of these core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family (see FIG. 2). Theselective proteolysis of synaptic SNAREs accounts for the total block ofneurotransmitter release caused by Clostridial toxins in vivo. The SNAREprotein targets of Clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); M. Zouhair Atassi,Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, (Dirk W.Dressler & Joseph J. Jankovic eds., 2003); Giovanna Lalli et al., TheJourney of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) TrendsMicrobiol. 431-437, (2003) which are hereby incorporated by reference.

TeNT and BoNT/B, /D, /F, and /G specifically recognize VAMP (also knownas synaptobrevin), an integral protein of the synaptic vesicle membrane.VAMP is cleaved at distinct bonds depending on the toxin. BoNT/A and /Erecognize and specifically cleave SNAP-25, a protein of the presynapticmembrane, at two different sites in the carboxy-terminal portion of theprotein. BoNT/C1 cleaves syntaxin, a protein of the nerve plasmalemma,in addition to SNAP-25. The three protein targets of the CoNTs areconserved from yeast to humans although cleavage sites and toxinsusceptibility are not necessarily conserved, see below; see, also,e.g., Humeau, supra, (2000); Heiner Niemann et al., Clostridialneurotoxins: new tools for dissecting exocytosis, 4(5) Trends Cell Biol.179-185 (1994); and Rossella Pellizzari et al., Tetanus and botulinumneurotoxins: mechanism of action and therapeutic uses, 354(1381) Philos.Trans. R. Soc. Lond. B Biol. Sci. 259-268 (1999).

The natural targets of the Clostridial toxins include VAMP, SNAP-25, andsyntaxin. VAMP is associated with the synaptic vesicle membrane, whereasSNAP-25 and syntaxin are associated with the plasma membrane (see FIG.3). BoNT/A and BoNT/E cleave SNAP-25 in the carboxy-terminal region,releasing a nine or twenty-six amino acid segment, respectively, andBoNT/C1 also cleaves SNAP-25 near the carboxy-terminus. The botulinumserotypes BoNT/B, BoNT/D, BoNT/F and BoNT/G, and tetanus toxin, act onthe conserved central portion of VAMP, and release the amino-terminalportion of VAMP into the cytosol. BoNT/C1 cleaves syntaxin at a singlesite near the cytosolic membrane surface. Thus, BoNT/B, BoNT/C1, BoNT/D,BoNT/F, BoNT/G or TeNT proteolysis results in release of a large portionof the cytosolic domain of VAMP or syntaxin, while only a small portionof SNAP-25 is released by BoNT/A, BoNT/C1 or BoNT/E cleavage, see, e.g.,Humeau et al., supra, (2000); Turton et al., supra, (2002); Lalli etal., supra (2003).

Naturally occurring SNAP-25, a protein of about 206 residues lacking atransmembrane segment, is associated with the cytosolic surface of thenerve plasmalemma (see FIG. 3). SNAP-25 is required for axonal growthduring development and may be required for nerve terminal plasticity inthe mature nervous system. SNAP-25 has been isolated from a variety ofvertebrate and invertebrate species including, e.g., species belongingto the genera Homo, Macaca, Bos, Rattus, Mus, Gallus, Carassius, Danio,Torpedo, Xenopus, Strongylocentrotus, Drosophila, Hirudo, Loligo,Lymnaea and Caenorhabditis. In humans, at least two isoforms aredifferentially expressed during development; isoform a is constitutivelyexpressed during fetal development, while isoform b appears at birth andpredominates in adult life. SNAP-25 analogues such as SNAP-23 also areexpressed outside the nervous system, for example, in pancreatic cells.

Naturally occurring VAMP is a protein of about 120 residues, with theexact length depending on the species and isoform. As shown in FIG. 3,VAMP contains a short carboxy-terminal segment inside the vesicle lumenwhile most of the molecule is exposed to the cytosol. The proline-richamino-terminal thirty residues are divergent among species and isoformswhile the central portion of VAMP (residues 30 to 96), which is rich incharged and hydrophilic residues and includes known cleavage sites, ishighly conserved. VAMP colocalizes with synaptophysin on synapticvesicle membranes. VAMP has been isolated from a variety of vertebrateand invertebrate species including, e.g., species belonging to thegenera Homo, Macaca, Bos, Rattus, Mus, Gallus, Danio, Torpedo, Xenopus,Strongylocentrotus, Drosophila, Hirudo, Loligo, Lymnaea, Aplysia andCaenorhabditis. In addition, multiple isoforms of VAMP have beenidentified including VAMP-1, VAMP-2 and VAMP-3/cellubrevin, and formsinsensitive to toxin cleavage have been identified in non-neuronalcells. VAMP appears to be present in all vertebrate tissues although thedistribution of VAMP-1 and VAMP-2 varies in different cell types.Chicken and rat VAMP-1 are not cleaved by TeNT or BoNT/B. These VAMP-1orthologs have a valine in place of the glutamine present in human andmouse VAMP-1 at the TeNT or BoNT/B cleavage site. The substitution doesnot affect BoNT/D, /F or /G, which cleave both VAMP-1 and VAMP-2 withsimilar rates.

Naturally occurring Syntaxin is located on the cytosolic surface of thenerve plasmalemma and is membrane-anchored via a carboxy-terminalsegment, with most of the protein exposed to the cytosol (see FIG. 3).Syntaxin colocalizes with calcium channels at the active zones of thepresynaptic membrane, where neurotransmitter release takes place. Inaddition, syntaxin interacts with synaptotagmin, a protein of the SSVmembrane that forms a functional bridge between the plasmalemma and thevesicles. Syntaxin has been isolated from a variety of vertebrate andinvertebrate species including, e.g., species belonging to the generaHomo, Bos, Rattus, Mus, Gallus, Danio, Strongylocentrotus, Drosophila,Hirudo, Loligo, Lymnaea and Aplysia. Three isoforms of slightlydifferent length (285 and 288 residues) have been identified in nervecells (isoforms 1A, 1B1 and 1B2), with isoforms 2, 3, 4 and 5 expressedin other tissues. The different isoforms have varying sensitivities toBoNT/C1, with the 1A, 1B, 1B2, 2 and 3 syntaxin isoforms cleaved by thistoxin.

Aspects of the present invention provide for compositions comprising anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of a cell. These substrates are comprised of afluorescent member, a membrane targeting domain and a Clostridial toxinrecognition sequence comprising a cleavage site, where the cleavage siteintervenes between the fluorescent member and the membrane localizationdomain.

The Clostridial toxin substrates disclosed in the present specificationinclude, in part, a Clostridial toxin recognition sequence including acleavage site. By definition, a Clostridial toxin substrate issusceptible to cleavage by at least one Clostridial toxin underconditions suitable for Clostridial toxin protease activity. A varietyof Clostridial toxin substrates are discussed herein below. AdditionalClostridial toxin substrates are described in, e.g., Lance E. Steward,et al., FRET Protease Assays for Clostridial Toxins, U.S. PatentPublication 2003/0143651 (Jul. 31, 2003); Lance E. Steward, et al., FRETProtease Assays for Botulinum Serotype A/E Toxins, U.S. PatentPublication 2003/0143650 (Jul. 31, 2003); and Ester Fernandez-Salas, etal., Cell-based Fluorescence Resonance Energy Transfer (FRET) Assays forClostridial Toxins, U.S. Patent Publication 2004/0072270 (Apr. 15,2004).

The Clostridial toxin substrates disclosed in the present specificationcomprise, in part, a Clostridial toxin recognition sequence including acleavage site. As used herein, the term “Clostridial toxin recognitionsequence” means a scissile bond together with adjacent or non-adjacentrecognition elements, or both, sufficient for detectable proteolysis atthe scissile bond by a Clostridial toxin under conditions suitable forClostridial toxin protease activity. A variety of Clostridial toxinrecognition sequences are discussed herein below.

Clostridial toxin substrates useful in aspects of the invention includepeptides and peptidomimetics as well as derivatized forms thereof. Asused herein, the term “peptidomimetic” is used broadly to mean apeptide-like molecule that is cleaved by the same Clostridial toxin asthe peptide substrate upon which it is structurally based. Suchpeptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,which are peptide-like molecules resulting from oligomeric assembly ofN-substituted glycines, and are cleaved by the same Clostridial toxin asthe peptide substrate upon which the peptidomimetic is derived (see, forexample, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger'sMedicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; JohnWiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an NC^(δ) or C^(α)-C^(δ) cyclizedamino acid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic which mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

In other embodiments, a Clostridial toxin substrate useful in theinvention is a peptide or peptidomimetic having a defined length. AClostridial toxin substrate can be, for example, a peptide orpeptidomimetic having at least 100, at least 150, at least 200, at least250, at least 300, at least 350 or at least 500 residues. In otherembodiments, a Clostridial toxin substrate has at most 20 residues, atmost 30 residues, at most 40 residues, at most 50 residues, at most 100residues, at most 150 residues, at most 200 residues, at most 250residues, at most 300 residues, at most 350 residues or at most 400residues.

A wide variety of Clostridial toxin recognition sequence are useful inaspects of the invention. Specific and distinct cleavage sites fordifferent Clostridial toxins are well known in the art. BoNT/A cleaves aGln-Arg bond; BoNT/B and TeNT cleave a Gln-Phe bond; BoNT/C1 cleaves aLys-Ala or Arg-Ala bond; BoNT/D cleaves a Lys-Leu bond; BoNT/E cleavesan Arg-Ile bond; BoNT/F cleaves a Gln-Lys bond; and BoNT/G cleaves anAla-Ala bond (see Table 1). In standard nomenclature, the sequencesurrounding a Clostridial toxin cleavage site is denotedP₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ with P₁-P₁′ representing the scissilebond. It is understood that a P₁ or P₁′ site, or both, can besubstituted with another amino acid or amino acid mimetic in place ofthe naturally occurring residue. As an example, BoNT/A substrates havebeen prepared in which the P₁ position (Gln) is modified to be analanine, 2-aminobutyric acid or asparagine residue; these substrateswere hydrolyzed by BoNT/A at the P₁-Arg bond, see, e.g., James J.Schmidt & Karen A Bostian, Endoproteinase activity of type A botulinumneurotoxin: substrate requirements and activation by serum albumin,16(1) J. Protein Chem. 19-26 (1997). While it is recognized thatsubstitutions can be introduced at the P₁ position of the scissile bond,for example, a BoNT/A scissile bond, it is further recognized thatconservation of the P₁′ residue can be advantageous, see, e.g.,Vadakkanchery V. Vaidyanathan et al., Proteolysis of SNAP-25 isoforms bybotulinum neurotoxin types A, C, and E: domains and amino acid residuescontrolling the formation of enzyme-substrate complexes and cleavage,72(1) J Neurochem. 327-337 (1999).

TABLE 1 Bonds Cleaved in Human VAMP-2, SNAP-25 or Syntaxin-1 ToxinTarget P₄₋P₃₋P₂₋P₁--P₁′-P₂′-P₃′-P₄′ SEQ ID NO: BoNT/A SNAP-25Glu-Ala-Asn-Gln-Arg*-Ala-Thr-Lys 96 BoNT/B VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser 97 BoNT/C1 Syntaxin-1Asp-Thr-Lys-Lys-Ala*-Val-Lys-Tyr 98 BoNT/C1 SNAP-25Ala-Asn-Gln-Arg-Ala*-Thr-Lys-Met 99 BoNT/D VAMP-2Arg-Asp-Gln-Lys-Leu*-Ser-Glu-Leu 100 BoNT/E SNAP-25Gln-Ile-Asp-Arg-Ile*-Met-Glu-Lys 101 BoNT/F VAMP-2Glu-Arg-Asp-Gln-Lys*-Leu-Ser-Glu 102 BoNT/G VAMP-2Glu-Thr-Ser-Ala-Ala*-Lys-Leu-Lys 103 TeNT VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser 104 *Scissile bond shown in bold

Thus, an embodiment, is a composition comprising an exogenousClostridial toxin substrate capable of being localized to the plasmamembrane of a cell wherein said substrate comprises a fluorescentmember, a membrane targeting domain and a Clostridial toxin recognitionsequence comprising a cleavage site, where the cleavage site intervenesbetween said fluorescent member and said membrane localization domain.In an aspect of this embodiment, a Clostridial toxin substrate comprisesa Clostridial toxin recognition sequence in which the P₁′ residue is notmodified or substituted relative to the naturally occurring residue in atarget protein cleaved by the Clostridial toxin. In another aspect ofthis embodiment, a Clostridial toxin substrate comprises a Clostridialtoxin recognition sequence in which the P₁ residue is modified orsubstituted relative to the naturally occurring residue in a targetprotein cleaved by the Clostridial toxin; such a Clostridial toxinsubstrate retains susceptibility to peptide bond cleavage between the P₁and P₁′ residues.

Any of a variety of Clostridial toxin recognition sequences are usefulin the cells of the invention including, without limitation, botulinumtoxin recognition sequences such as BoNT/A recognition sequences, BoNT/Brecognition sequences, BoNT/C1 recognition sequences, BoNT/D recognitionsequences, BoNT/E recognition sequences, BoNT/F recognition sequences,BoNT/G recognition sequences and TeNT recognition sequences.

A variety of BoNT/A recognition sequences are well known in the art andare useful in the invention, see, e.g., Mark A. Breidenbach & Axel T.Brunger, Substrate recognition strategy for botulinum neurotoxinserotype A, 432(7019) Nature 925-929 (2004). A BoNT/A recognitionsequence can have, for example, residues 46-206, residues 134 to 206,residues 137 to 206 or 146-206 of human SNAP-25, see, e.g., Teresa A.Ekong et al., Recombinant SNAP-25 is an effective substrate forClostridium botulinum type A toxin endopeptidase activity in vitro, 143(Pt 10) Microbiology 3337-3347 (1997); Clifford C. Shone et al., ToxinAssays, U.S. Pat. No. 5,962,637 (Oct. 5, 1999); and Vaidyanathan et al.,supra, (1999). A BoNT/A recognition sequence also can include, withoutlimitation, the sequenceThr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 105) ora peptidomimetic thereof, which corresponds to residues 190 to 202 ofhuman SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO:106) or a peptidomimetic thereof, which corresponds to residues 187 to201 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ IDNO: 107) or a peptidomimetic thereof, which corresponds to residues 187to 202 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQID NO: 108) or a peptidomimetic thereof, which corresponds to residues187 to 203 of human SNAP-25;Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQID NO: 109) or a peptidomimetic thereof, which corresponds to residues186 to 202 of human SNAP-25; orAsp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu(SEQ ID NO: 110) or a peptidomimetic thereof, which corresponds toresidues 186 to 203 of human SNAP-25. See, for example, James J. Schmidt& Karen A Bostian, Proteolysis of synthetic peptides by type A botulinumneurotoxin, 14(8) J. Protein Chem. 703-708 (1995); Schmidt & Bostian,supra, (1997); James J. Schmidt et al., Type A botulinum neurotoxinproteolytic activity: development of competitive inhibitors andimplications for substrate specificity at the S1′ binding subsite,435(1) FEBS Lett. 61-64 (1998); and James J. Schmidt & Karen A Bostian,Assay for the proteolytic activity of serotype a from clostridiumbotulinum, U.S. Pat. No. 5,965,699 (Oct. 12, 1999).

A BoNT/A recognition sequence useful in aspects of the invention cancorrespond to a segment of a protein that is sensitive to cleavage bybotulinum toxin serotype A, or can be substantially similar to a segmentof a BoNT/A-sensitive protein. As shown in Table 2, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/A are knownin the art and include, for example, human, rat, mouse, Danio,Carassius, SNAP-25A and SNAP-25B; and Torpedo SNAP-25. Thus, a BoNT/Arecognition sequence can correspond, for example, to a segment of humanSNAP-25A or SNAP-25B; bovine SNAP-25A or SNAP-25B; rat SNAP-25A orSNAP-25B; mouse SNAP-25A or SNAP-25B; Xenopus SNAP-25A or SNAP-25B;Danio SNAP-25A or SNAP-25B; Carassius SNAP-25A or SNAP-25B; TorpedoSNAP-25; Strongylocentrotus SNAP-25; Loligo SNAP-25; Lymnaea SNAP-25;Aplysia SNAP-25, isoforms thereof, or another naturally occurringprotein sensitive to cleavage by BoNT/A. Furthermore, comparison ofnative SNAP-25 amino acid sequences cleaved by BoNT/A reveals that suchsequences are not absolutely conserved (see Table 2), indicating that avariety of amino acid substitutions and modifications relative to anaturally occurring BoNT/A-sensitive SNAP-25 sequence can be toleratedin a BoNT/A recognition sequence useful in the invention. It isunderstood that a similar BoNT/A recognition sequence can be prepared,if desired, from a corresponding (homologous) segment of anotherBoNT/A-sensitive SNAP-25 isoform, paralog or ortholog, such as, theBoNT/A recognition sequence contain in the SNAP-25 proteins identifiedin the organisms listed above and in Table 2.

TABLE 2 Cleavage of SNAP-25 and Related Proteins^(a,b,c) Cleavage SitesOrganism Isoform BoNT/E BoNT/A BoNT/C1 Cleaved Susceptibility PrimateSNAP-25A MALDMGNEIDTQNRQIDR * IMEKADSNKTRIDEANQ * R * ATKMLGSG BoNT/A;BoNT/C1; BoNT/E SNAP-25B Primate SNAP-23A MALNIGNEIDAQN

Q

R — ITDKADTNRDRIDIAN

— R — AKKLIDS None^(b) SNAP-23B Rodent SNAP-25A MALDMGNEIDTQNRQIDR *IMEKADSNKTRIDEANQ * R * ATKMLGSG BoNT/A; BoNT/C1; BoNT/E SNAP-25B RodentSNAP-23 MALDMGNEIDAQNQQIQ

* ITEKADTNKNRIDIAN

— R — AKKLIDS BoNT/E Bird SNAP-25B MALDMGNEIDTQNRQIDR *IMEKADSNKTRIDEANQ — R — ATKMLGSG BoNT/E Amphibian SNAP-25AMALDMGNEIDTQNRQIDR ND IMEKADSNKARIDEAN

ND

ND ATKMLGSG ND SNAP-25B Amphibian SNAP-23 MAIDMGNELESHNQQIGR NDINEKAETNKTRIDEAN

ND K ND AKKLIE ND Fish SNAP-25A MALDMGNEIDTQNRQIDR * IMEKADSNKTRIDEANQ *R * ATKMLGSG BoNT/A; BoNT/C1; BoNT/E SNAP-25B MALDMGNEIDTQNRQIDR *IMDMADSNKTRIDEANQ * R * ATKMLGSG Fish SNAP-23 LALDMGNEIDKQNKTIDR NDITDKADMNKARIDEANQ ND R ND ANKLL ND Ray SNAP-25 MALDMSNEIGSQNAQIDR  —^(c)IV

KGDMNKARIDEAN

*

ND ATKML BoNT/A Sea urchin SNAP-25 MAIDMQSEIGAQNSQVGR NDITSKAESNEGRINSAD

ND R ND AKNILRNK ND Insect SNAP-25 MALDMGSELENQNRQIDR —INRKGESNEARIAVANQ — R * AHQLLK BoNT/C1 Insect SNAP-24 MALDMGSELENQNKQVDRND INAKGDANNIRMDGVN

ND R ND ANNLLKS ND Segmented SNAP-25 MAVDMGSEIDSQNRQVDR NDINNKMTSNQLRISDAN

— R ND ASKLLKE ND worm Cephalopod SNAP-25 MAIDMGNEIGSQNRQVDR NDIQQKAESNESRIDEAN

ND

ND ATKLLKN ND Gastropod SNAP-25 MAVDMGNEIESQNKQLDR ND INQKGGSLNVRVDEAN

ND R ND ANRILRKQ ND Round SNAP-25 MAIDMSTEVSNQNRQLDR * IHDKAQSNEVRVESAN

— R — AKNLITK BoNT/E worm Proteolytic cleavage occurs at this site (*);Proteolytic cleavage not detected at this site (—); Proteolytic cleavagenot determined at this site (ND) ^(a)= In vitro cleavage of SNAP-25requires 1000-fold higher BoNT/C concentration than BoNT/A or /E. ^(b)=Substitution of P182R, or K185DD (boxes) induces susceptibility towardBoNT/E. ^(c)= Resistance to BoNT/E possibly due to D189 or E189substitution by V189, see box.

Table 2—Cleavage of SNAP-25 and related proteins. Primate: HumanSNAP-25A residues 163-206 of SEQ ID NO: 1; Human SNAP-25B residues163-206 of SEQ ID NO: 2; Human SNAP-23A residues 169-211 of SEQ ID NO:3; Human SNAP-23B residues 116-158 of SEQ ID NO: 4; Monkey SNAP-25Bresidues 163-206 of SEQ ID NO: 5; Rodent: Rat SNAP-25A residues 163-206of SEQ ID NO: 6; Rat SNAP-25B residues 163-206 of SEQ ID NO: 7; MouseSNAP-25B residues 163-206 of SEQ ID NO: 8; Rat SNAP-23 residues 168-210of SEQ ID NO: 9; Mouse SNAP-23 residues 168-210 of SEQ ID NO: 10; Bird:Chicken SNAP-25B residues 163-206 of SEQ ID NO: 11; Fish: GoldfishSNAP-25A residues 161-204 of SEQ ID NO: 12; Goldfish SNAP-25B residues160-203 of SEQ ID NO: 13; Zebrafish SNAP-25A residues 161-204 of SEQ IDNO: 14; Zebrafish SNAP-25B residues 160-203 of SEQ ID NO: 15; ZebrafishSNAP-23 residues 174-214 of SEQ ID NO: 16; Ray: marbled electric raySNAP-25 residues 170-210 of SEQ ID NO: 17; Amphibian: Frog SNAP-25Aresidues 163-206 of SEQ ID NO: 18; Frog SNAP-25B residues 163-206 of SEQID NO: 19; Frog SNAP-23 residues 163-204 of SEQ ID NO: 20; Sea urchinSNAP-25 residues 169-212 of SEQ ID NO: 21; Insect: Fruit fly SNAP-25residues 171-212 of SEQ ID NO: 22 212; Fruit fly SNAP-24 residues170-212 of SEQ ID NO: 23; Segmented worm: Leech SNAP-25 residues 170-212of SEQ ID NO: 24; Cephalopod: squid SNAP-25 residues 245-267 of SEQ IDNO: 25; Gastropod: Pond snail SNAP-25 residues 244-266 of SEQ ID NO: 26;Round worm: Nematode worm SNAP-25 residues 165-207 of SEQ ID NO: 27.

A Clostridial toxin substrate, such as a substrate containing a BoNT/Arecognition sequence, can have one or multiple modifications as comparedto a naturally occurring sequence that is cleaved by the correspondingClostridial toxin. As an example, as compared to a 17-mer correspondingto residues 187 to 203 of human SNAP-25, substitution of Asp193 with Asnin the BoNT/A substrate resulted in a relative rate of proteolysis of0.23; substitution of Glu194 with Gln resulted in a relative rate of2.08; substitution of Ala195 with 2-aminobutyric acid resulted in arelative rate of 0.38; and substitution of Gln197 with Asn,2-aminobutyric acid or Ala resulted in a relative rate of 0.66, 0.25, or0.19, respectively (see Table 3). Furthermore, substitution of Ala199with 2-aminobutyric acid resulted in a relative rate of 0.79;substitution of Thr200 with Ser or 2-aminobutyric acid resulted in arelative rate of 0.26 or 1.20, respectively; substitution of Lys201 withAla resulted in a relative rate of 0.12; and substitution of Met202 withAla or norleucine resulted in a relative rate of 0.38 or 1.20,respectively, see, e.g., Schmidt & Bostian, supra, (1997). These resultsindicate that a variety of residues can be substituted in a Clostridialtoxin substrate as compared to a naturally occurring toxin-sensitivesequence. In the case of BoNT/A, these results indicate that residuesincluding but not limited to Glu194, Ala195, Gln197, Ala199, Thr200 andMet202, Leu203, Gly204, Ser205, and Gly206, as well as residues moredistal from the Gln-Arg scissile bond, can be substituted or conjugatedto a fluorophore, bulking group, donor fluorophore or acceptor in aBoNT/A substrate useful in the invention. Such a BoNT/A substrate isdetectably proteolyzed at the scissile bond by BoNT/A under conditionssuitable for Clostridial toxin protease activity. Thus, a BoNT/Asubstrate can include, if desired, one or several amino acidsubstitutions, additions or deletions relative to a naturally occurringSNAP-25 sequence.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Asubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/A recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. As used herein, the term“botulinum toxin serotype A recognition sequence” is synonymous with“BoNT/A recognition sequence” and means a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by a BoNT/A under conditionssuitable for Clostridial toxin protease activity. A scissile bondcleaved by BoNT/A can be, for example, Gln-Arg.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/A recognition sequence comprising a BoNT/Arecognition sequence containing at least six consecutive residues ofSNAP-25 including Gln-Arg. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/A recognitionsequence comprising the BoNT/A recognition sequenceGlu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO: 96). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/A recognition sequence comprising a portion of SNAP-25 such as,e.g., residues 1 to 206 of SEQ ID NO: 1; residues 46 to 206 of SEQ IDNO: 1; residues 134 to 206 of SEQ ID NO: 1; residues 137 to 206 of SEQID NO: 1; residues 146 to 206 of SEQ ID NO: 1, or a peptidomimeticthereof. In still other aspects of this embodiment, the Clostridialtoxin substrate includes, in part, a BoNT/A recognition sequencecomprising SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:108, SEQ ID NO: 109, or SEQ ID NO: 110, or a peptidomimetic thereof.

TABLE 3 Kinetic Parameters of BoNT/A Synthetic Peptide SubstratesRelative Peptide Sequence^(a) SEQ ID NO: Rate^(b) [1-15] SNKTRIDEANQRATK106 0.03 [1-16] SNKTRIDEANQRATKM 107 1.17 [1-17] SNKTRIDEANQRATKML 1081.00 M16A SNKTRIDEANQRATK A L 111 0.38 M16X SNKTRIDEANQRATK X L 112 1.20K15A SNKTRIDEANQRAT A ML 113 0.12 T14S SNKTRIDEANQRA S KML 114 0.26 T14BSNKTRIDEANQRA B KML 115 1.20 A13B SNKTRIDEANQR B TKML 116 0.79 Q11ASNKTRIDEAN A RATKML 117 0.19 Q11B SNKTRIDEAN B RATKML 118 0.25 Q11NSNKTRIDEAN N RATKML 119 0.66 N10A SNKTRIDEA A QRATKML 120 0.06 A9BSNKTRIDE B NQRATKML 121 0.38 E8Q SNKTRID Q ANQRATKML 122 2.08 D7N SNKTRIN EANQRATKML 123 0.23 ^(a)Nonstandard abbreviations: B, 2-aminobutyricacid; X, 2-aminohexanoic acid (norleucine) ^(b)Initial hydrolysis ratesrelative to peptide [1-17]. Peptide concentrations were 1.0 mM.

A variety of BoNT/B recognition sequences are well known in the art orcan be defined by routine methods. Such BoNT/B recognition sequences caninclude, for example, a sequence corresponding to some or all of thehydrophilic core of a VAMP protein such as human VAMP-1 or human VAMP-2.A BoNT/B recognition sequence can include, without limitation, residues33 to 94, residues 45 to 94, residues 55 to 94, residues 60 to 94,residues 65 to 94, residues 60 to 88 or residues 65 to 88 of humanVAMP-2 (SEQ ID NO: 31), or residues 60 to 94 of human VAMP-1-1 (SEQ IDNO: 28), VAMP-1-2 (SEQ ID NO: 29) and VAMP-1-3 (SEQ ID NO: 30) see,e.g., Shone et al., Eur. J. Biochem. 217: 965-971 (1993); and Shone etal., supra, (Oct. 5, 1999). A BoNT/B recognition sequence also caninclude, without limitation, the sequenceLeu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser-Gln-Phe-Glu-Thr-Ser-Ala-Ala-Lys-Leu-Lys-Arg-Lys-Tyr-Trp-Trp-Lys-Asn-Leu-Lys(SEQ ID NO: 124) or a peptidomimetic thereof, which corresponds toresidues 60 to 94 of human VAMP-2, see, e.g., James J. Schmidt & RobertG. Stafford, High Throughput Assays for the Proteolytic Activities ofClostridial Neurotoxins, U.S. Pat. No. 6,762,280 (Jul. 13, 2004) and theBoNT/B recognition sequenceLeu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser-Gln-Phe-Glu-Ser-Ser-Ala-Ala-Lys-Leu-Lys-Arg-Lys-Tyr-Trp-Trp-Lys-Asn-Cys-Lys(SEQ ID NO: 125) or a peptidomimetic thereof, which corresponds toresidues 62 to 96 of human VAMP-1.

A BoNT/B recognition sequence useful in aspects of the invention cancorrespond to a segment of a protein that is sensitive to cleavage bybotulinum toxin serotype B, or can be substantially similar to a segmentof a BoNT/B-sensitive protein. As shown in Table 4, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/B are knownin the art and include, for example, human and mouse VAMP-1, VAMP-2 andVAMP-3/cellubrevin; bovine VAMP-2; rat VAMP-2 and VAMP-3; chickenVAMP-2; Torpedo VAMP-1; Strongylocentrotus VAMP; Drosophila sybA, synB,synC, synD and synE; Hirudo VAMP; and Caenorhabditis SNB1-like. Thus, aBoNT/B recognition sequence can correspond, for example, to a segment ofhuman VAMP-1, VAMP-2 or VAMP-3; bovine VAMP-2; rat VAMP-2 or VAMP-3;mouse VAMP-1, VAMP-2 or VAMP-3; chicken VAMP-1, VAMP-2 or VAMP-3;Xenopus VAMP-2 or VAMP-3; Danio VAMP-1 or VAMP-2; Torpedo VAMP-1;Strongylocentrotus VAMP; Drosophila sybA, synB, synC, synD or synE;Hirudo VAMP; Loligo VAMP; Lymnaea VAMP; Aplysia VAMP; CaenorhabditisSNB1, isoforms thereof, or another naturally occurring protein sensitiveto cleavage by BoNT/B. Furthermore, as shown in Table 4, comparison ofnative VAMP amino acid sequences cleaved by BoNT/B reveals that suchsequences are not absolutely conserved, indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring VAMP sequence can be tolerated in a BoNT/B substrate of theinvention. It is understood that a similar BoNT/B recognition sequencecan be prepared, if desired, from a corresponding (homologous) segmentof another BoNT/B-sensitive VAMP-1 or VAMP-2 isoform, paralog orortholog, such as, the BoNT/B recognition sequence contain in the VAMP-1and VAMP-2 proteins identified in the organisms listed above and inTable 4.

TABLE 4 Cleavage of VAMP and Related Proteins Cleavage Sites TeNTOrganism Isoform BoNT/F BoNT/D BoNT/B BoNT/G Cleaved SusceptibilityPrimate VAMP1-1 RVNVDKVLERDQ * K * LSELDDRADALQAGASQ * FESSA * AKLKRKYWWBoNT/B; BoNT/D; BoNT/F; VAMP1-2 BoNT/G; TeNT VAMP1-3 Primate VAMP2RVNVDKVLERDQ * K * LSELDDRADALQAGASQ * FETSA * AKLKRKYWW BoNT/B; BoNT/D;BoNT/F; BoNT/G; TeNT Primate VAMP3 RVNVDKVLERDQ * K *LSELDDRADALQAGASQ * FETSA * AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; BoNT/G;TeNT Bovine VAMP2 RVNVDKVLERDQ * K * LSELDDRADALQAGASQ * FETSA *AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; BoNT/G; TeNT Rodent VAMP1/1bRVNVDKVLERDQ * K * LSELDDRADALQAGAS

 —^(a) FESSA * AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; VAMP1 RVNVDKVLERDQ *K * LSELDDRADALQAGASQ * FESSA * AKLKRKYWW BoNT/G; TeNT Rodent VAMP2RVNVDKVLERDQ * K * LSELDDRADALQAGASQ * FETSA * AKLKRKYWW BoNT/B; BoNT/D;BoNT/F; VAMP2-b BoNT/G; TeNT Rodent VAMP3 RVNVDKVLERDQ * K *LSELDDRADALQAGASQ * FETSA * AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; BoNT/G;TeNT Bird VAMP1 RVNVDKVLERDQ * K * LSELDDRADALQAGAS

* FESSA * AKLKRKYWW BoNT/D; BoNT/F; BoNT/G Bird VAMP2 RMNVDKVLERDQ * K *LSELDNRADALQAGASQ * FETSA * AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; BoNT/G;TeNT Bird VAMP3 RVNVDKVLERDQ ND K ND LSELDDRADALQAGASQ ND FETSA NDAKLKRKYWW ND Amphibian VAMP2 RVNVDKVLERD

ND K ND LSELDDRADALQAGASQ ND FETSA ND AKLKRKYWW ND Amphibian VAMP3RVNVDKVLERDQ ND K ND LSELDDRADALQAGASQ ND FETSA ND AKLKRKYWW ND FishVAMP1 RVNVDKVLERDQ ND K ND LSELDDRADALQAGASQ ND FESSA ND AKLKNKYWW NDFish VAMP2 RVNVDKVLERDQ ND K ND LSELDDRADALQAGASQ ND FETSA ND AKLKNKYWWND Fish VAMP-3 RVNVDKVLERDQ ND K ND LSELDDRADALQAGASQ ND FETSA NDAKLKRKYWW ND Ray VAMP1 RVNVDKVLERDQ * K * LSELDDRADALQAGASQ * FESSA *AKLKRKYWW BoNT/B; BoNT/D; BoNT/F; BoNT/G; TeNT Sea urchin VAMPRVNVDKVLERDQ —

— LSVLDDRADALQQGASQ * FETNA —

KLKRKYWW BoNT/B; TeNT Insect Syn-A1 RVNVEKVLERDQ * K *LSELGERADQLEQGASQ * FEQQA —

KLKRKQWW BoNT/B; BoNT/D; BoNT/F; Syn-B1 TeNT Insect Syn-A2RVNVEKVLERDQ * K * LSELGERADQLEQGASQ —

EQQA —

KLKRKQWW BoNT/D; BoNT/F Syn-B2 Insect Syn-C RTNVEKVLERD

— K * LSELDDRADALQQGASQ * FEQQA —

KLKRKFWL BoNT/B; BoNT/D; TeNT Syn-D Syn-E Segmented VAMP RVNVDKVLEKDQ *K * LAELDGRADALQAGASQ * FEASA —

KLKRKFWW BoNT/B; BoNT/D; BoNT/F; worm TeNT Cephalopod VAMP RVNVDKVLERD

ND K ND

SELDDRADALQAGASQ ND FEASA ND

KLKRKFWW ND Gastropod VAMP RVNVEKVLDRDQ ND K ND

SQLDDRAEALQAGASQ ND FEASA ND

KLKRKYWW ND Round SNB1 KVNVEKVLERDQ ND K ND LSQLDDRADALQEGASQ ND FEKSAND ATLKRKYWW BoNT/B; TeNT worm SNB-like RNNVNKVMERD

—

— LNSLDHRAEVLQNGASQ * FQQS

—

TLRQKYWW Proteolytic cleavage occurs at this site (*); Proteolyticcleavage not detected at this site (—); Proteolytic cleavage notdetermined at this site (ND) ^(a)= Rat VAMP1 resistance to BoNT/B andTeNT possibly due to Q189V substitution, see box.

Table 4—Cleavage of VAMP and related proteins. Primate: Human VAMP-1-1residues 49-92 of SEQ ID NO: 28; Human VAMP-1-2 residues 49-92 of SEQ IDNO: 29; Human VAMP-1-3 residues 49-92 of SEQ ID NO: 30; Human VAMP-2residues 47-90 of SEQ ID NO: 31; Monkey VAMP-2 residues 47-90 of SEQ IDNO: 32; Human VAMP-3/cellubrevin residues 30-73 of SEQ ID NO: 33;Bovine: Cow VAMP-2 residues 47-90 of SEQ ID NO: 34; Rodent: Rat VAMP-1residues 49-92 of SEQ ID NO: 35; Rat VAMP-1-b residues 49-92 of SEQ IDNO: 36; Mouse VAMP-1 residues 49-92 of SEQ ID NO: 37; Rat VAMP-2residues 47-90 of SEQ ID NO: 38; Rat VAMP-2-b residues 47-90 of SEQ IDNO: 39; Mouse VAMP-2 residues 47-90 of SEQ ID NO: 40; RatVAMP-3/cellubrevin residues 34-77 of SEQ ID NO: 41; MouseVAMP-3/cellubrevin residues 34-77 of SEQ ID NO: 42; Bird: Chicken VAMP-1residues 190-233 of SEQ ID NO: 43; Chicken VAMP-2 residues 47-88 of SEQID NO: 44; Chicken VAMP-3/cellubrevin residues 34-77 of SEQ ID NO: 45;Fish: Zebrafish VAMP-1 residues 50-93 of SEQ ID NO: 46; Zebrafish VAMP-2residues 41-84 of SEQ ID NO: 47; Zebrafish VAMP-3 residues 33-60 of SEQID NO: 48; Ray: marbled electric ray VAMP-1 residues 51-94 of SEQ ID NO:49; Amphibian: Frog VAMP-2 residues 45-88 of SEQ ID NO: 50; Frog VAMP-3residues 32-75 of SEQ ID NO: 51; Sea urchin VAMP residues 31-74 of SEQID NO: 52; Insect: Fruit fly SynA1 residues 40-83 of SEQ ID NO: 53;Fruit fly SynA2 residues 63-106 of SEQ ID NO: 54; Fruit fly SynB1residues 63-106 of SEQ ID NO: 55; Fruit fly SynB2 residues 63-106 of SEQID NO: 56; Fruit fly SynC residues 57-100 of SEQ ID NO: 57; Fruit flySynD residues 66-109 of SEQ ID NO: 58; Fruit fly SynE residues 57-100 ofSEQ ID NO: 59; Segmented worm: Leech VAMP residues 45-88 of SEQ ID NO:60; Cephalopod: squid VAMP residues 56-99 of SEQ ID NO: 61; Gastropod:Pond snail VAMP residues 49-92 of SEQ ID NO: 62; sea hare VAMP residues37-80 of SEQ ID NO: 63; Round worm: Nematode worm SNB1 residues 72-115of SEQ ID NO: 64; Nematode worm SNB-like residues 82-115 of SEQ ID NO:65.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Bsubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/B recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. As used herein, the term“botulinum toxin serotype B recognition sequence” is synonymous with“BoNT/B recognition sequence” and means a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by a BoNT/B underappropriate conditions. A scissile bond cleaved by BoNT/B can be, forexample, Gln-Phe.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/B recognition sequence comprising a BoNT/Brecognition sequence containing at least six consecutive residues ofVAMP including Gln-Phe. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/B recognitionsequence comprising the BoNT/B recognition sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 97). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/B recognition sequence comprising a portion of VAMP-1-1 such as,e.g., residues 1 to 118 of SEQ ID NO: 28; residues 62 to 96 of SEQ IDNO: 28, or a peptidomimetic thereof. In other aspects of thisembodiment, the Clostridial toxin substrate includes, in part, a BoNT/Brecognition sequence comprising a portion of VAMP-1-2 such as, e.g.,residues 1 to 117 of SEQ ID NO: 29; residues 62 to 96 of SEQ ID NO: 29,or a peptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/B recognitionsequence comprising a portion of VAMP-1-3 such as, e.g., residues 1 to116 of SEQ ID NO: 30; residues 62 to 96 of SEQ ID NO: 30, or apeptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/B recognitionsequence comprising a portion of VAMP-2 such as, e.g., residues 1 to 116of SEQ ID NO: 31; residues 33 to 94 of SEQ ID NO: 31; residues 45 to 94of SEQ ID NO: 31; residues 55 to 94 of SEQ ID NO: 31; residues 60 to 94of SEQ ID NO: 31; residues 65 to 94 of SEQ ID NO: 31; residues 60 to 88of SEQ ID NO: 31; residues 65 to 88 of SEQ ID NO: 31, or apeptidomimetic thereof.

It is understood that a BoNT/C1 recognition sequence can correspond to asegment of a protein that is sensitive to cleavage by botulinum toxinserotype C1, or can be substantially similar to a segment of aBoNT/C1-sensitive protein. As further shown in Table 5, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/C1 are knownin the art and include, for example, human and mouse Syntaxin 1A,Syntaxin 1B1 and Syntaxin 1B2; bovine and rat Syntaxin 1A and Syntaxin1B2; rat Syntaxin 2 and Rat syntaxin 3; Strongylocentrotus Syntaxin;Drosophila Syntaxin 1A; Hirudo Syntaxin 1A; Loligo Syntaxin 1A; AplysiaSyntaxin 1A. Thus, a BoNT/C1 recognition sequence can correspond, forexample, to a segment of human Syntaxin 1A, Syntaxin 1B1, Syntaxin 1B2,Syntaxin 2-1, Syntaxin 2-2, Syntaxin 2-3 or Syntaxin 3A; bovine Syntaxin1A, Syntaxin 1B1 or Syntaxin 1B2; rat Syntaxin 1A, Syntaxin 1B1,Syntaxin 1B2, Syntaxin 2 or Syntaxin 3A; mouse Syntaxin 1A, Syntaxin1B1, Syntaxin 1B2, Syntaxin 2, Syntaxin 3A, Syntaxin 3B or Syntaxin 3C;chicken Syntaxin 1A or Syntaxin 2; Xenopus Syntaxin 1A or Syntaxin 1B;Danio Syntaxin 1A, Syntaxin 1B or Syntaxin 3; Torpedo Syntaxin 1A orSyntaxin 1B; Strongylocentrotus Syntaxin 1A or Syntaxin 1B; DrosophilaSyntaxin 1A or Syntaxin 1B; Hirudo Syntaxin 1A or Syntaxin 1B; LoligoSyntaxin 1A or Syntaxin 1B; Lymnaea Syntaxin 1A or Syntaxin 1B, isoformsthereof, or another naturally occurring protein sensitive to cleavage byBoNT/C1. Furthermore, comparison of native syntaxin amino acid sequencescleaved by BoNT/C1 reveals that such sequences are not absolutelyconserved (see Table 5), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive syntaxin sequence can be tolerated in a BoNT/C1substrate useful in the invention. It is understood that a similarBoNT/C1 recognition sequence can be prepared, if desired, from acorresponding (homologous) segment of another BoNT/C1-sensitive syntaxinisoform, paralog or ortholog, such as, the BoNT/C1 recognition sequencecontain in the syntaxin proteins identified in the organisms listedabove and in Table 5.

Table 5—Cleavage of Syntaxin and related proteins. Primate: HumanSyntaxin1A residues 242-264 of SEQ ID NO: 66; Human Syntaxin1B1 residues241-263 of SEQ ID NO: 67; Human Syntaxin1B2 residues 241-263 of SEQ IDNO: 68; Human Syntaxin2-1 residues 241-263 of SEQ ID NO: 69; HumanSyntaxin2-2 residues 241-263 of SEQ ID NO: 70; Human Syntaxin2-3residues 241-263 of SEQ ID NO: 71; Human Syntaxin3 residues 241-263 ofSEQ ID NO: 72; Bovine: Cow Syntaxin1A residues 242-264 of SEQ ID NO: 73;Cow Syntaxin1B2 residues 241-263 of SEQ ID NO: 74; Rodent: RatSyntaxin1A residues 242-264 of SEQ ID NO: 75; Rat Syntaxin1B2 residues241-263 of SEQ ID NO: 76; Mouse Syntaxin1A residues 242-264 of SEQ IDNO: 77; Mouse Syntaxin1B1 residues 241-263 of SEQ ID NO: 78; MouseSyntaxin1B2 residues 241-263 of SEQ ID NO: 79; Rat Syntaxin2 residues243-265 of SEQ ID NO: 80; Mouse Syntaxin2 residues 242-264 of SEQ ID NO:81; Rat Syntaxin3A residues 241-263 of SEQ ID NO: 82; Mouse Syntaxin3Aresidues 241-263 of SEQ ID NO: 83; Mouse Syntaxin3B residues 241-263 ofSEQ ID NO: 84; Mouse Syntaxin3C residues 223-245 of SEQ ID NO: 85; Bird:Chicken Syntaxin1B residues 235-257 of SEQ ID NO: 86; Chicken Syntaxin2residues 240-262 of SEQ ID NO: 87; Fish: Zebrafish Syntaxin1B residues241-263 of SEQ ID NO: 88; Zebrafish Syntaxin3 residues 239-261 of SEQ IDNO: 89; sea urchin Syntaxin1B residues 241-263 of SEQ ID NO: 90; Insect:Fruit fly Syntaxin1A residues 245-267 of SEQ ID NO: 91; Segmented worm:leech Syntaxin1A residues 248-270 of SEQ ID NO: 92; Cephalopod: squidSyntaxin1A residues 245-267 of SEQ ID NO: 93; Gastropod: Pond snailSyntaxin1A residues 244-266 of SEQ ID NO: 94; sea hare Syntaxin1Aresidues 244-266 of SEQ ID NO: 95.

TABLE 5 Cleavage of Syntaxin and Related Proteins Cleavage Site CleavedOrganism Isoform BoNT/C1 Susceptibility Primate Syntaxin1ADYVERAVSDTKK * AVKYQSKARRK BoNT/C1 Syntaxin1B1 Syntaxin1B2 PrimateSyntaxin2-1 DYVEHAKEETKK ND AIKYQSKARRK ND Syntaxin2-2 Syntaxin2-3Primate Syntaxin3A DHVEKARDESKK ND AVKYQSQARKK ND Bovine Syntaxin1ADYVERAVSDTKK * AVKYQSKARRK BoNT/C1 SyntaxinB2 Rodent Syntaxin1ADYVERAVSDTKK * AVKYQSKARRK BoNT/C1 Syntaxin1B1 Syntaxin1B2 RodentSyntaxin2 DYVEHAKEETKK * AIKYQSKARRK BoNT/C1 Rodent Syntaxin3ADHVEKARDETK

* AMKYQGQARKK BoNT/C1 Rodent Syntaxin3B GFVERAVADTKK ND AVKYQSEARRK NDSyntaxin3C Bird Syntaxin1B DYVEPWFVTK

ND AVMYQCKSRRK ND Bird Syntaxin2 DYVEHAKEETKK ND AVKYQSKARRK ND FishSyntaxin1B DYVERAVSDTKK * AVKYQSQARKK BoNT/C1 Fish Syntaxin3DHVEAARDETKK ND AVRYQSKARKK ND Sea urchin Syntaxin1B DYVRRQNDTKK *AVKYQSKARRK BoNT/C1 Insect Syntaxin1A DYVQTATQDTKK * ALKYQSKARRK BoNT/C1Segmented Syntaxin1A DYVETAAADTKK * AMKYQSAARKK BoNT/C1 worm CephalopodSyntaxin1A DYIETAKVDTKK * AVKYQSKARQK BoNT/C1 Gastropod Syntaxin1ADYIETAKMDTKK * AVKYQSKARRK BoNT/C1 Proteolytic cleavage occurs at thissite (*); Proteolytic cleavage not detected at this site (—);Proteolytic cleavage not determined at this site (ND)

As further shown in Table 2, a variety of naturally occurring proteinssensitive to cleavage by BoNT/C1 are known in the art and include, forexample, human, rat, mouse, Danio, Carassius SNAP-25A and SNAP-25B; andDrosophila SNAP-25. Thus, a BoNT/C1 recognition sequence can correspond,for example, to a segment of human SNAP-25A or SNAP-25B; bovine SNAP-25Aor SNAP-25B; rat SNAP-25A or SNAP-25B; mouse SNAP-25A or SNAP-25B;Xenopus SNAP-25A or SNAP-25B; Danio SNAP-25A or SNAP-25B; CarassiusSNAP-25A or SNAP-25B; Torpedo SNAP-25; Strongylocentrotus SNAP-25;Drosophila SNAP-25 or SNAP-24; Hirudo SNAP-25; Loligo SNAP-25; LymnaeaSNAP-25, isoforms thereof, or another naturally occurring proteinsensitive to cleavage by BoNT/C1. As discussed above in regard tovariants of naturally occurring syntaxin sequences, comparison of nativeSNAP-25 amino acid sequences cleaved by BoNT/C1 reveals significantsequence variability (Table 2), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive SNAP-25 sequence can be tolerated in a BoNT/C1substrate useful in the invention. It is understood that a similarBoNT/C1 recognition sequence can be prepared, if desired, from acorresponding (homologous) segment of another BoNT/C1-sensitive SNAP-25isoform, paralog or ortholog, such as, the BoNT/A recognition sequencecontain in the SNAP-25 proteins identified in the organisms listed aboveand in Table 2.

Thus, in an embodiment, a composition comprises an exogenous BoNT/C1substrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/C1 recognition sequence comprising acleavage site, where the cleavage site intervenes between saidfluorescent member and said membrane localization domain. As usedherein, the term “botulinum toxin serotype C1 recognition sequence” issynonymous with “BoNT/C1 recognition sequence” and means a scissile bondtogether with adjacent or non-adjacent recognition elements, or both,sufficient for detectable proteolysis at the scissile bond by a BoNT/C1under appropriate conditions. A scissile bond cleaved by BoNT/C1 can be,for example, Lys-Ala or Arg-Ala.

In an aspect of this embodiment, the encoded Clostridial toxin substrateincludes, in part, a BoNT/C1 recognition sequence comprising a BoNT/C1recognition sequence containing at least six consecutive residues ofSyntaxin including Lys-Ala. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/C1 recognitionsequence comprising the BoNT/C1 recognition sequenceAsp-Thr-Lys-Lys-Ala-Val-Lys-Tyr (SEQ ID NO: 98). In another aspect ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/C1 recognition sequence comprising a BoNT/C1 recognition sequencecontaining at least six consecutive residues of SNAP-25 includingArg-Ala. In another aspect of this embodiment, the Clostridial toxinsubstrate includes, in part, a BoNT/C1 recognition sequence comprisingthe BoNT/C1 recognition sequence Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ IDNO: 99). In yet another aspect of this embodiment, the Clostridial toxinsubstrate includes, in part, a BoNT/C1 recognition sequence comprising aBoNT/C1 recognition sequence containing at least six consecutiveresidues of Syntaxin including Lys-Ala and a BoNT/C1 recognitionsequence comprising a BoNT/C1 recognition sequence containing at leastsix consecutive residues of SNAP-25 including Arg-Ala.

In other aspects of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/C1 recognition sequence comprising a portionof Syntaxin-1A such as, e.g., residues 1 to 288 of SEQ ID NO: 66, or apeptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/C1 recognitionsequence comprising a portion of Syntaxin-1B1 such as, e.g., residues 1to 288 of SEQ ID NO: 67, or a peptidomimetic thereof. In other aspectsof this embodiment, the Clostridial toxin substrate includes, in part, aBoNT/C1 recognition sequence comprising a portion of Syntaxin-1B2 suchas, e.g., residues 1 to 288 of SEQ ID NO: 68, or a peptidomimeticthereof. In other aspects of this embodiment, the Clostridial toxinsubstrate includes, in part, a BoNT/C1 recognition sequence comprising aportion of Syntaxin 2-1 such as, e.g., residues 1 to 287 of SEQ ID NO:69, or a peptidomimetic thereof. In other aspects of this embodiment,the Clostridial toxin substrate includes, in part, a BoNT/C1 recognitionsequence comprising a portion of Syntaxin-2-2 such as, e.g., residues 1to 288 of SEQ ID NO: 70, or a peptidomimetic thereof. In other aspectsof this embodiment, the Clostridial toxin substrate includes, in part, aBoNT/C1 recognition sequence comprising a portion of Syntaxin-2-3 suchas, e.g., residues 1 to 289 of SEQ ID NO: 71, or a peptidomimeticthereof. In other aspects of this embodiment, the Clostridial toxinsubstrate includes, in part, a BoNT/C1 recognition sequence comprising aportion of Syntaxin-3A such as, e.g., residues 1 to 289 of SEQ ID NO:83, or a peptidomimetic thereof. In other aspects of this embodiment,the Clostridial toxin substrate includes, in part, a BoNT/C1 recognitionsequence comprising a portion of Syntaxin-3B such as, e.g., residues 1to 283 of SEQ ID NO: 84, or a peptidomimetic thereof. In other aspectsof this embodiment, the Clostridial toxin substrate includes, in part, aBoNT/C1 recognition sequence comprising a portion of Syntaxin-3C suchas, e.g., residues 1 to 269 of SEQ ID NO: 85, or a peptidomimeticthereof.

In other aspects of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/C1 recognition sequence comprising a portionof SNAP-25 such as, e.g., residues 1 to 206 of SEQ ID NO: 1; residues 93to 206 of SEQ ID NO: 1; residues 134 to 206 of SEQ ID NO: 1; residues137 to 206 of SEQ ID NO: 1; residues 146 to 206 of SEQ ID NO: 1;residues 137 to 202 of SEQ ID NO: 1, or a peptidomimetic thereof. Instill other aspects of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/C1 recognition sequence comprising SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, orSEQ ID NO: 110, or a peptidomimetic thereof.

A variety of BoNT/D recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/D recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2, see, e.g.,Shinji Yamasaki et al., Cleavage of members of the synaptobrevin/VAMPfamily by types D and F botulinum neurotoxins and tetanus toxin, 269(17)J. Biol. Chem. 12764-12772 (1994). Thus, a BoNT/D recognition sequencecan include, for example, residues 27 to 69 or residues 37 to 69 of ratVAMP-2. A BoNT/D recognition sequence also can include, withoutlimitation, the sequenceAla-Gln-Val-Asp-Glu-Val-Val-Asp-Ile-Met-Arg-Val-Asn-Val-Asp-Lys-Val-Leu-Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser(SEQ ID NO: 126) or a peptidomimetic thereof, which corresponds toresidues 37 to 75 of human VAMP-2, see, e.g., Schmidt & Stafford, supra,(Jul. 13, 2004) and the BoNT/D recognition sequenceAla-Gln-Val-Glu-Glu-Val-Val-Asp-Ile-Ile-Arg-Val-Asn-Val-Asp-Lys-Val-Leu-Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser(SEQ ID NO: 127) or a peptidomimetic thereof, which corresponds toresidues 39 to 77 of the human VAMP-1 isoforms, VAMP-1-1, VAMP-1-2 andVAMP-1-3.

A BoNT/D recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype D, or can besubstantially similar to a segment of a BoNT/D-sensitive protein. Asshown in Table 4, a variety of naturally occurring proteins sensitive tocleavage by BoNT/D are known in the art and include, for example, human,rat and mouse VAMP-1, VAMP-2 and VAMP-3/cellubrevin; bovine VAMP-2;chicken VAMP-1, VAMP-2 and VAMP-3; Xenopus VAMP-2 or VAMP-3; DanioVAMP-1 or VAMP-2; Torpedo VAMP-1; Strongylocentrotus VAMP; DrosophilasybA, synB, synC, synD, synE; Hirudo VAMP; Loligo VAMP; Lymnaea VAMP;Aplysia VAMP; and Caenorhabditis SNB1. Thus, a BoNT/D recognitionsequence can correspond, for example, to a segment of human VAMP-1,VAMP-2 or VAMP-3; bovine VAMP-2; rat VAMP-1, VAMP-2 or VAMP-3; mouseVAMP-1, VAMP-2 or VAMP-3; chicken VAMP-1, VAMP-2 or VAMP-3; XenopusVAMP-2 or VAMP-3; Danio VAMP-1 or VAMP-2; Torpedo VAMP-1;Strongylocentrotus VAMP; Drosophila sybA, synB, synC, synD, synE; HirudoVAMP; Loligo VAMP; Lymnaea VAMP; Aplysia VAMP; Caenorhabditis SNB1,isoforms thereof, or another naturally occurring protein sensitive tocleavage by BoNT/D. Furthermore, as shown in Table 4 above, comparisonof native VAMP amino acid sequences cleaved by BoNT/D revealssignificant sequence variability, indicating that a variety of aminoacid substitutions and modifications relative to a naturally occurringBoNT/D-sensitive VAMP sequence can be tolerated in a BoNT/D substrateuseful in the invention. It is understood that a similar BoNT/Drecognition sequence can be prepared, if desired, from a corresponding(homologous) segment of another BoNT/D-sensitive VAMP-1 or VAMP-2isoform, paralog or ortholog, such as, the BoNT/B recognition sequencecontain in the VAMP-1 and VAMP-2 proteins identified in the organismslisted above and in Table 4.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Dsubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/D recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. The term “botulinum toxinserotype D recognition sequence” is synonymous with “BoNT/D recognitionsequence” and means a scissile bond together with adjacent ornon-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by a BoNT/D under appropriateconditions. A scissile bond cleaved by BoNT/D can be, for example,Lys-Leu.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/D recognition sequence comprising a BoNT/Drecognition sequence containing at least six consecutive residues ofVAMP including Lys-Leu. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/D recognitionsequence comprising the BoNT/D recognition sequenceArg-Asp-Gln-Lys-Leu-Ser-Glu-Leu (SEQ ID NO: 100). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/D recognition sequence comprising a portion of VAMP-1-1 such as,e.g., residues 1 to 118 of SEQ ID NO: 28; residues 39 to 77 of SEQ IDNO: 28, or a peptidomimetic thereof. In other aspects of thisembodiment, the Clostridial toxin substrate includes, in part, a BoNT/Drecognition sequence comprising a portion of VAMP-1-2 such as, e.g.,residues 1 to 117 of SEQ ID NO: 29; residues 39 to 77 of SEQ ID NO: 29,or a peptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/D recognitionsequence comprising a portion of VAMP-1-3 such as, e.g., residues 1 to116 of SEQ ID NO: 30; residues 39 to 77 of SEQ ID NO: 30, or apeptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/D recognitionsequence comprising a portion of VAMP-2 such as, e.g., residues 1 to 116of SEQ ID NO: 31; residues 1 to 86 of SEQ ID NO: 31; residues 1 to 76 ofSEQ ID NO: 31; residues 1 to 69 of SEQ ID NO: 31; residues 27 to 116 ofSEQ ID NO: 31; residues 37 to 116 of SEQ ID NO: 31; residues 27 to 68 ofSEQ ID NO: 31; residues 37 to 69 of SEQ ID NO: 31, or a peptidomimeticthereof.

One skilled in the art appreciates that a BoNT/E recognition sequencecan correspond to a segment of a protein that is sensitive to cleavageby botulinum toxin serotype E, or can be substantially similar to asegment of a BoNT/E-sensitive protein. A BoNT/E recognition sequence canhave, for example, residues 46-206, residues 92 to 206, residues,residues 134 to 206, residues, 137 to 206; 146-206 or 156-206 of humanSNAP-25, see, e.g., Vaidyanathan et al., supra, (1999); and Schmidt &Stafford, supra, (Jul. 13, 2004).

A BoNT/E recognition sequence useful in aspects of the invention cancorrespond to a segment of a protein that is sensitive to cleavage bybotulinum toxin serotype E, or can be substantially similar to a segmentof a BoNT/E-sensitive protein. As shown in Table 2, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/E are knownin the art and include, for example, human, chicken, Danio, CarassiusSNAP-25A and SNAP-25B; rat and mouse SNAP-25A, SNAP-25B and SNAP-23; andCaenorhabditis SNAP-25. Thus, a BoNT/E recognition sequence cancorrespond, for example, to a segment of human SNAP-25A or SNAP-25B;bovine SNAP-25A or SNAP-25B; rat SNAP-25A, SNAP-25B or SNAP-23; mouseSNAP-25A, SNAP-25B or SNAP-23; Xenopus SNAP-25A or SNAP-25B; DanioSNAP-25A or SNAP-25B; Carassius SNAP-25A or SNAP-25B; StrongylocentrotusSNAP-25; Drosophila SNAP-24; Hirudo SNAP-25; Loligo SNAP-25; LymnaeaSNAP-25; Caenorhabditis SNAP-25, isoforms thereof, or another naturallyoccurring protein sensitive to cleavage by BoNT/C1. Furthermore, asshown in Table 2, comparison of native SNAP-23 and SNAP-25 amino acidsequences cleaved by BoNT/E reveals that such sequences are notabsolutely conserved, indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/E-sensitive SNAP-23 or SNAP-25 sequence can be tolerated in aBoNT/E substrate useful in the invention. It is understood that asimilar BoNT/E recognition sequence can be prepared, if desired, from acorresponding (homologous) segment of another BoNT/E-sensitive SNAP-25isoform, paralog or ortholog, such as, the BoNT/E recognition sequencecontain in the SNAP-25 proteins identified in the organisms listed aboveand in Table 2.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Esubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/E recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. As used herein, the term“botulinum toxin serotype E recognition sequence” is synonymous with“BoNT/E recognition sequence” and means a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by a BoNT/E underappropriate conditions. A scissile bond cleaved by BoNT/E can be, forexample, Arg-Ile.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/E recognition sequence comprising a BoNT/Erecognition sequence containing at least six consecutive residues ofSNAP-25 including Arg-Ile. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/E recognitionsequence comprising the BoNT/E recognition sequenceGln-Ile-Asp-Arg-Ile-Met-Glu-Lys (SEQ ID NO: 101). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/E recognition sequence comprising a portion of SNAP-25 such as,e.g., residues 1 to 206 of SEQ ID NO: 1; residues 46 to 206 of SEQ IDNO: 1; residues 92 to 206 of SEQ ID NO: 1; residues 134 to 206 of SEQ IDNO: 1; residues 137 to 206 of SEQ ID NO: 1, residues 146 to 206 of SEQID NO: 1; residues 156 to 206 of SEQ ID NO: 1, or a peptidomimeticthereof.

A variety of BoNT/F recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/F recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2, see, e.g.,Yamasaki et al., supra, (1994). A BoNT/F recognition sequence also caninclude, for example, residues 27 to 69 or residues 37 to 69 of ratVAMP-2. It is understood that a similar BoNT/F recognition sequence canbe prepared, if desired, from a corresponding (homologous) segment ofanother BoNT/F-sensitive VAMP isoform, paralog or ortholog, such as,e.g., human VAMP-1 or human VAMP-2. A BoNT/F recognition sequence alsocan include, without limitation, the sequenceAla-Gln-Val-Asp-Glu-Val-Val-Asp-Ile-Met-Arg-Val-Asn-Val-Asp-Lys-Val-Leu-Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser(SEQ ID NO: 126) or a peptidomimetic thereof, which corresponds toresidues 37 to 75 of human VAMP-2, see, e.g., Schmidt & Stafford, supra,(Jul. 13, 2004) and the BoNT/F recognition sequenceAla-Gln-Val-Glu-Glu-Val-Val-Asp-Ile-Ile-Arg-Val-Asn-Val-Asp-Lys-Val-Leu-Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu-Leu-Asp-Asp-Arg-Ala-Asp-Ala-Leu-Gln-Ala-Gly-Ala-Ser(SEQ ID NO: 127) or a peptidomimetic thereof, which corresponds toresidues 39 to 77 of human VAMP-1.

A BoNT/F recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype F, or can besubstantially similar to a segment of a BoNT/F-sensitive protein. Asshown in Table 4, a variety of naturally occurring proteins sensitive tocleavage by BoNT/F are known in the art and include, for example, human,rat and mouse VAMP-1, VAMP-2 and VAMP-3/cellubrevin; bovine VAMP-2;chicken VAMP-1 and VAMP-2; Torpedo VAMP-1; and Drosophila sybA and synB.Thus, a BoNT/F recognition sequence can correspond, for example, to asegment of human VAMP-1, VAMP-2 or VAMP-3; bovine VAMP-2; rat VAMP-1,VAMP-2 or VAMP-3; mouse VAMP-1, VAMP-2 or VAMP-3; chicken VAMP-1, VAMP-2or VAMP-3; Xenopus VAMP-2 or VAMP-3; Danio VAMP-1 or VAMP-2; TorpedoVAMP-1; Drosophila sybA and synB; Hirudo VAMP; Loligo VAMP; LymnaeaVAMP; Aplysia VAMP; Caenorhabditis SNB1, isoforms thereof, or anothernaturally occurring protein sensitive to cleavage by BoNT/F. Thus, aBoNT/F recognition sequence can correspond, for example, to a segment ofhuman VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2,rat VAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2, TorpedoVAMP-1, Aplysia VAMP, Drosophila syb, leech VAMP, or another naturallyoccurring protein sensitive to cleavage by BoNT/F. Furthermore, as shownin Table 4 above, comparison of native VAMP amino acid sequences cleavedby BoNT/F reveals that such sequences are not absolutely conserved,indicating that a variety of amino acid substitutions and modificationsrelative to a naturally occurring BoNT/F-sensitive VAMP sequence can betolerated in a BoNT/F substrate useful in the invention. It isunderstood that a similar BoNT/F recognition sequence can be prepared,if desired, from a corresponding (homologous) segment of anotherBoNT/F-sensitive VAMP-1 or VAMP-2 isoform, paralog or ortholog, such as,the BoNT/F recognition sequence contain in the VAMP-1 and VAMP-2identified in the organisms listed above and in Table 4.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Fsubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/F recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. The term “botulinum toxinserotype F recognition sequence,” as used herein, is synonymous with“BoNT/F recognition sequence” and means a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by a BoNT/F underappropriate conditions. A scissile bond cleaved by BoNT/F can be, forexample, Gln-Lys.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/F recognition sequence comprising a BoNT/Frecognition sequence containing at least six consecutive residues ofVAMP including Gln-Lys. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/F recognitionsequence comprising the BoNT/F recognition sequenceGlu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 102). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/F recognition sequence comprising a portion of VAMP-1-1 such as,e.g., residues 1 to 118 of SEQ ID NO: 28; residues 39 to 77 of SEQ IDNO: 28, or a peptidomimetic thereof. In other aspects of thisembodiment, the Clostridial toxin substrate includes, in part, a BoNT/Frecognition sequence comprising a portion of VAMP-1-2 such as, e.g.,residues 1 to 117 of SEQ ID NO: 29; residues 39 to 77 of SEQ ID NO: 29,or a peptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/F recognitionsequence comprising a portion of VAMP-1-3 such as, e.g., residues 1 to116 of SEQ ID NO: 30; residues 39 to 77 of SEQ ID NO: 30, or apeptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/F recognitionsequence comprising a portion of VAMP-2 such as, e.g., residues 1 to 116of SEQ ID NO: 31; residues 1 to 86 of SEQ ID NO: 31; residues 1 to 76 ofSEQ ID NO: 31; residues 1 to 69 of SEQ ID NO: 31; residues 27 to 116 ofSEQ ID NO: 31; residues 37 to 116 of SEQ ID NO: 31; residues 27 to 68 ofSEQ ID NO: 31; residues 37 to 75 of SEQ ID NO: 31; residues 37 to 69 ofSEQ ID NO: 31, or a peptidomimetic thereof.

A BoNT/G recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype G, or can besubstantially similar to such a BoNT/G-sensitive segment. As shown inTable 4, a variety of naturally occurring proteins sensitive to cleavageby BoNT/G are known in the art and include, for example, human, rat andmouse VAMP-1, VAMP-2 and VAMP-3/cellubrevin; bovine VAMP-2; chickenVAMP-1, and VAMP-2; and Torpedo VAMP-1. Thus, a BoNT/G recognitionsequence can correspond, for example, to a segment of human VAMP-1,VAMP-2 or VAMP-3; bovine VAMP-2; rat VAMP-1, VAMP-2 or VAMP-3; mouseVAMP-1, VAMP-2 or VAMP-3; chicken VAMP-1, VAMP-2 or VAMP-3; XenopusVAMP-2 or VAMP-3; Danio VAMP-1 or VAMP-2; Torpedo VAMP-1; CaenorhabditisSNB1, isoforms thereof, or another naturally occurring protein sensitiveto cleavage by BoNT/G. Furthermore, as shown in Table 4 above,comparison of native VAMP amino acid sequences cleaved by BoNT/G revealsthat such sequences are not absolutely conserved, indicating that avariety of amino acid substitutions and modifications relative to anaturally occurring BoNT/G-sensitive VAMP sequence can be tolerated in aBoNT/G substrate useful in the invention. It is understood that asimilar BoNT/G recognition sequence can be prepared, if desired, from acorresponding (homologous) segment of another BoNT/G-sensitive VAMP-1 orVAMP-2 isoform, paralog or ortholog, such as, the BoNT/G recognitionsequence contain in the VAMP-1 and VAMP-2 identified in the organismslisted above and in Table 4.

Thus, in an embodiment, a composition comprises an exogenous BoNT/Gsubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a BoNT/G recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. As used herein, the term“botulinum toxin serotype G recognition sequence” is synonymous with“BoNT/G recognition sequence” and means a scissile bond together withadjacent or non-adjacent recognition elements, or both, sufficient fordetectable proteolysis at the scissile bond by a BoNT/G underappropriate conditions. A scissile bond cleaved by BoNT/G can be, forexample, Ala-Ala.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/G recognition sequence comprising a BoNT/Grecognition sequence containing at least six consecutive residues ofVAMP including Ala-Ala. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/G recognitionsequence comprising the BoNT/G recognition sequenceGlu-Thr-Ser-Ala-Ala-Lys-Leu-Lys (SEQ ID NO: 103). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/G recognition sequence comprising a portion of VAMP-1-1 such as,e.g., residues 1 to 118 of SEQ ID NO: 28, or a peptidomimetic thereof.In other aspects of this embodiment, the Clostridial toxin substrateincludes, in part, a BoNT/G recognition sequence comprising a portion ofVAMP-1-2 such as, e.g., residues 1 to 117 of SEQ ID NO: 29, or apeptidomimetic thereof. In other aspects of this embodiment, theClostridial toxin substrate includes, in part, a BoNT/G recognitionsequence comprising a portion of VAMP-1-3 such as, e.g., residues 1 to116 of SEQ ID NO: 30, or a peptidomimetic thereof. In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aBoNT/G recognition sequence comprising a portion of VAMP-2 such as,e.g., residues 1 to 116 of SEQ ID NO: 31, or a peptidomimetic thereof.

A variety of TeNT recognition sequences are well known in the art or canbe defined by routine methods and include sequences corresponding tosome or all of the hydrophilic core of a VAMP protein such as humanVAMP-1 or human VAMP-2. A TeNT recognition sequence can include, forexample, residues 25 to 93 or residues 33 to 94 of human VAMP-2 (SEQ IDNO: 31; F. Cornille et al., Solid-phase synthesis, conformationalanalysis and in vitro cleavage of synthetic human synaptobrevin II 1-93by tetanus toxin L chain, 222(1) Eur. J. Biochem. 173-181 (1994);Patrick Foran et al., Differences in the protease activities of tetanusand botulinum B toxins revealed by the cleavage of vesicle-associatedmembrane protein and various sized fragments, 33(51) Biochemistry15365-15374 (1994); residues 51 to 93 or residues 1 to 86 of rat VAMP-2,see, e.g., Yamasaki et al., supra, (1994); or residues 33 to 94 of humanVAMP-1-1 (SEQ ID NO: 28), residues 33 to 94 of human VAMP-1-2 (SEQ IDNO: 29) and residues 33 to 94 of human VAMP-1-3 (SEQ ID NO: 30). A TeNTrecognition sequence also can include, for example, residues 25 to 86,residues 33 to 86 or residues 51 to 86 of human VAMP-2 (SEQ ID NO: 31)or rat VAMP-2 (SEQ ID NO: 38). It is understood that a similar TeNTrecognition sequence can be prepared, if desired, from a corresponding(homologous) segment of another TeNT-sensitive VAMP isoform or specieshomolog such as human VAMP-1 or sea urchin or Aplysia VAMP.

Thus, a TeNT recognition sequence can correspond to a segment of aprotein that is sensitive to cleavage by tetanus toxin, or can besubstantially similar to a segment of a TeNT-sensitive protein. As shownin Table 4, a variety of naturally occurring proteins sensitive tocleavage by TeNT are known in the art and include, for example, humanand mouse VAMP-1, VAMP-2 and VAMP-3/cellubrevin; bovine VAMP-2; ratVAMP-2 and VAMP-3; chicken VAMP-2; Torpedo VAMP-1; StrongylocentrotusVAMP; Drosophila sybA, synB, synC, synD and synE; Hirudo VAMP; andCaenorhabditis SNB1-like. Thus, a TeNT recognition sequence cancorrespond, for example, to a segment of human VAMP-1, VAMP-2 or VAMP-3;bovine VAMP-2; rat VAMP-2 or VAMP-3; mouse VAMP-1, VAMP-2 or VAMP-3;chicken VAMP-1, VAMP-2 or VAMP-3; Xenopus VAMP-2 or VAMP-3; Danio VAMP-1or VAMP-2; Torpedo VAMP-1; Strongylocentrotus VAMP; Drosophila sybA,synB, synC, synD or synE; Hirudo VAMP; Loligo VAMP; Lymnaea VAMP;Aplysia VAMP; Caenorhabditis SNB1 and SNB-like, isoforms thereof, oranother naturally occurring protein sensitive to cleavage by TeNT.Furthermore, comparison of native VAMP amino acid sequences cleaved byTeNT reveals that such sequences are not absolutely conserved (Table 4).This finding indicates that a variety of amino acid substitutions andmodifications relative to a naturally occurring TeNT-sensitive VAMPsequence can be tolerated in a TeNT substrate useful in the invention.It is understood that a similar TeNT recognition sequence can beprepared, if desired, from a corresponding (homologous) segment ofanother TeNT-sensitive VAMP-1 or VAMP-2 isoform, paralog or ortholog,such as, the TeNT recognition sequence contain in the VAMP-1 and VAMP-2identified in the organisms listed above and in Table 4.

Thus, in an embodiment, a composition comprises an exogenous TeNTsubstrate capable of being localized to the plasma membrane of a cellwherein said substrate comprises a fluorescent member, a membranetargeting domain and a TeNT recognition sequence comprising a cleavagesite, where the cleavage site intervenes between said fluorescent memberand said membrane localization domain. As used herein, the term “tetanustoxin recognition sequence” means a scissile bond together with adjacentor non-adjacent recognition elements, or both, sufficient for detectableproteolysis at the scissile bond by a tetanus toxin under appropriateconditions. A scissile bond cleaved by TeNT can be, for example,Gln-Phe.

In an aspect of this embodiment, the Clostridial toxin substrateincludes, in part, a TeNT recognition sequence comprising a TeNTrecognition sequence containing at least six consecutive residues ofVAMP including Gln-Phe. In another aspect of this embodiment, theClostridial toxin substrate includes, in part, a TeNT recognitionsequence comprising the TeNT recognition sequenceGly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 104). In other aspects ofthis embodiment, the Clostridial toxin substrate includes, in part, aTeNT recognition sequence comprising a portion of VAMP-1-1 such as,e.g., residues 1 to 118 of SEQ ID NO: 28 or residues 33 to 94 of SEQ IDNO: 28. In other aspects of this embodiment, the Clostridial toxinsubstrate includes, in part, a TeNT recognition sequence comprising aportion of VAMP-1-2 such as, e.g., residues 1 to 117 of SEQ ID NO: 29 orresidues 33 to 94 of SEQ ID NO: 29. In other aspects of this embodiment,the Clostridial toxin substrate includes, in part, a TeNT recognitionsequence comprising a portion of VAMP-1-3 such as, e.g., residues 1 to116 of SEQ ID NO: 30 or residues 33 to 94 of SEQ ID NO: 30. In otheraspects of this embodiment, the Clostridial toxin substrate includes, inpart, a TeNT recognition sequence comprising a portion of VAMP-2 suchas, e.g., residues 1 to 116 of SEQ ID NO: 31; residues 25 to 94 of SEQID NO: 31; residues 33 to 94 of SEQ ID NO: 31; residues 51 to 93 of SEQID NO: 31; residues 1 to 86 of SEQ ID NO: 31; residues 25 to 86 of SEQID NO: 31; residues 33 to 86 of SEQ ID NO: 31; residues 51 to 86 of SEQID NO: 31, or a peptidomimetic thereof.

SNAP-25, VAMP and Syntaxin share a short motif usually located withinregions predicted to adopt an α-helical conformation called the SNAREmotif. This motif usually comprises a nine amino acid motif with thegeneral formula of H-Θ-Θ-X-H-Θ-X-H-P (see FIG. 2 b), where H is aaliphatic residue, Θ is a carboxylate residue, P is a polar residue andX is any amino acid, see e.g., Ornella Rossetto et al., SNARE motif andneurotoxins, 372(6505) Nature 415-416 (1994); Rossella Pellizzari etal., Structural determinants of the specificity for synapticvesicle-associated membrane protein/synaptobrevin of tetanus andbotulinum type B and G neurotoxins, 271(34) J. Biol. Chem. 20353-20358(1996); Rossella Pellizzari et al., The interaction of synapticvesicle-associated membrane protein/synaptobrevin with botulinumneurotoxins D and F, 409(3) FEBS Lett. 339-342 (1997); and PhilipWashbourne et al., Botulinum neurotoxin types A and E require the SNAREmotif in SNAP-25 for proteolysis, 418(1-2) FEBS Lett. 1-5 (1997). Thismotif is present in SNAP-25, VAMP and syntaxin isoforms expressed inanimals sensitive to the toxins. In contrast, Drosophila and yeastSNAP-25 proteins are resistant to these toxins. In addition, VAMP andsyntaxin isoforms not involved in exocytosis contain sequence variationsin these α-helical motif regions.

Multiple repetitions of the α-helical motif are present in proteinssensitive to cleavage by Clostridial toxins: Four copies are naturallypresent in SNAP-25, designated S1-S4; two copies are naturally presentin VAMP, designated V1 and V2; and two copies are naturally present insyntaxin, designated X1 and X2, see, e.g., Humeau et al., supra, (2000).Furthermore, peptides corresponding to the specific sequence of theα-helical motifs can inhibit toxin activity in vitro and in vivo, andsuch peptides can cross-inhibit different toxins. In addition,antibodies raised against such peptides can cross-react among the threetarget proteins, indicating that this α-helical motif is exposed on theprotein surface and adopts a similar configuration in each of the threetarget proteins. Consistent with these findings, SNAP-25-specific,VAMP-specific and syntaxin-specific toxins cross-inhibit each other bycompeting for the same binding site, although they do not cleave targetsnon-specifically. These results indicate that a Clostridial toxinrecognition sequence can include, if desired, at least one α-helicalmotif. It is recognized that an α-helical motif is not required forcleavage by a Clostridial toxin, as evidenced by 16-mer and 17-mersubstrates for BoNT/A known in the art, see, e.g., Schmidt & Bostian,supra, (1997); Schmidt & Bostian, supra, (Oct. 12, 1999); and Schmidt &Stafford, supra, (Jul. 13, 2004).

Although multiple α-helical motifs are found in the naturally occurringSNAP-25, VAMP and syntaxin target proteins, a Clostridial toxinrecognition sequence useful in a Clostridial toxin substrate can have asingle α-helical motif. In particular embodiments, a method of theinvention relies on a Clostridial toxin recognition sequence includingtwo or more α-helical motifs. A BoNT/A or BoNT/E recognition sequencecan include, for example, the S4 α-helical motif, alone or combined withone or more additional α-helical motifs; a BoNT/B, BoNT/G or TeNTrecognition sequence can include, for example, the V2 α-helical motif,alone or combined with one or more additional α-helical motifs; aBoNT/C1 recognition sequence can include, for example, the S4 α-helicalmotif, alone or combined with one or more additional α-helical motifs,or the X2 α-helical motif, alone or combined with one or more additionalα-helical motifs; and a BoNT/D or BoNT/F recognition sequence caninclude, for example, the V1 α-helical motif, alone or combined with oneor more additional α-helical motifs. Representative SNARE motifs arepresented in Tables 6, 7 and 8.

TABLE 6 SNARE motifs of SNAP-25 and Related Proteins Motif OrganismIsoform S1 S2 S3 S4 Primate SNAP-25A ADESLESTR VEESKDAGI LDEQGEQLDMDENLEQVS SNAP-25B LDEQGEQLE Primate SNAP-23A TDESLESTR AIESQDAGILDEQKEQLN MEENLTQVG SNAP-23B Rodent SNAP-25A ADESLESTR VEESKDAGILDEQGEQLD MDENLEQVS SNAP-25B LDEQGEQLE Rodent SNAP-23 TDESLESTRAIESQDAGI LDEQGEQLN MEENLTQVG Bird SNAP-25B ADESLESTR VEESKDAGILDEQGEQLE MDENLEQVS Amphibian SNAP-25A ADESLESTR VEGSKDAGI LDEQGEQLDMDENLEQVG SNAP-25B LDEQGEQLE Amphibian SNAP-23 ADESLESTR ALESQDAGILDEQGEQLD MDENLVQVG Fish SNAP-25A ADESLESTR VEESKDAGI LDEQGEQLEMDENLEQVG SNAP-25B GDESLESTR MDENLEQVG Fish SNAP-23 TDESLESTR AEESRETGVLDEQGEQLR MEENLDQVG Ray SNAP-25 TDESLESTR VEESKDAGI LDEQGEQLE MEENLDQVGSea urchin SNAP-25 TDESLESTR AEESKEAGI LDEQGEQLD MDENLTQVS InsectSNAP-25 ADESLESTR CEESKEAGI LDDQGEQLD MEENMGQVN Insect SNAP-24 ADESLESTRMDESKEAGI LDDQGEQLD MDENLGQVN Segmented worm SNAP-25 TDDSLESTR CEESKDAGILDEQGEQLD MEQNMGEVS Cephalopod SNAP-25 TDDSLESTR CEESKEAGI LDEQGEQLDMENNMKEVS Gastropod SNAP-25 TNESLESTR CEESKEAGI LDEQGEQLD MEQNIGEVARound worm SNAP-25 TDDSLESTR CEESKEAGI LDDQGEQLE MDENVQQVS Proteolyticcleavage occurs at this site (*); Proteolytic cleavage not detected atthis site (—); Proteolytic cleavage not determined at this site (ND)

Table 6—SNARE motifs of SNAP-25 and Related Proteins. Primate: HumanSNAP-25A residues 22-30, 36-44, 50-58 and 146-154 of SEQ ID NO: 1; HumanSNAP-25B residues 22-30, 36-44, 50-58 and 146-154 of SEQ ID NO: 2; HumanSNAP-23A residues 17-25, 31-39, 45-53, and 152-160 of SEQ ID NO: 3;Human SNAP-23B residues 17-25, 31-39, 45-53 and 152-160 of SEQ ID NO: 4;Monkey SNAP-25B residues 22-30, 36-44, 50-58 and 146-154 of SEQ ID NO:5; Rodent: Rat SNAP-25A residues 22-30, 36-44, 50-58 and 146-154 of SEQID NO: 6; Rat SNAP-25B residues 22-30, 36-44, 50-58 and 146-154 of SEQID NO: 7; Mouse SNAP-25B residues 22-30, 36-44, 50-58 and 146-154 of SEQID NO: 8; Rat SNAP-23 residues 17-25, 31-39, 45-53 and 151-159 of SEQ IDNO: 9; Mouse SNAP-23 residues 17-25, 31-39, 45-53 and 151-159 of SEQ IDNO: 10; Bird: Chicken SNAP-25B residues 22-30, 36-44, 50-58 and 146-154of SEQ ID NO: 11; Fish: Goldfish SNAP-25A residues 22-30, 36-44, 50-58and 144-152 of SEQ ID NO: 12; Goldfish SNAP-25B residues 22-30, 36-44,50-58 and 143-151 of SEQ ID NO: 13; Zebrafish SNAP-25A residues 22-30,36-44, 50-58 and 144-152 of SEQ ID NO: 14; Zebrafish SNAP-25B residues22-30, 36-44, 50-58 and 143-151 of SEQ ID NO: 15; Zebrafish SNAP-23residues 17-25, 31-39, 45-53 and 157-165 of SEQ ID NO: 16; Ray: marbledelectric ray SNAP-25 residues 26-34, 40-48, 54-62 and 153-161 of SEQ IDNO: 17; Amphibian: Frog SNAP-25A residues 22-30, 36-44, 50-58 and146-154 of SEQ ID NO: 18; Frog SNAP-25B residues 22-30, 36-44, 50-58 and146-154 of SEQ ID NO: 19; Frog SNAP-23 residues 17-25, 31-39, 45-53 and146-154 of SEQ ID NO: 20; Sea urchin SNAP-25 residues 24-32, 38-46,52-60 and 152-160 of SEQ ID NO: 21; Insect: Fruit fly SNAP-25 residues29-37, 43-51, 57-65 and 154-163 of SEQ ID NO: 22 212; Fruit fly SNAP-24residues 24-32, 38-46, 52-60 and 153-162 of SEQ ID NO: 23; Segmentedworm: Leech SNAP-25 residues 30-38, 44-52, 58-66 and 153-161 of SEQ IDNO: 24; Cephalopod: squid SNAP-25 residues 25-33, 39-47, 53-61 and153-161 of SEQ ID NO: 25; Gastropod: Pond snail SNAP-25 residues 32-40,46-54, 60-68 and 160-168 of SEQ ID NO: 26; Round worm: Nematode wormSNAP-25 residues 22-30, 36-44, 50-58 and 148-156 of SEQ ID NO: 27.

TABLE 7 SNARE motifs of VAMP and Related Proteins Motif Organism IsoformV1 V2 Primate VAMP1-1 VEEVVDIIR LDDRADALQ VAMP1-2 VAMP1-3 Primate VAMP2VDEVVDIMR LDDRADALQ Primate VAMP3 VDEVVDIMR LDDRADALQ Bovine VAMP2VDEVVDIMR LDDRADALQ Rodent VAMP1 VEEVVDIIR LDDRADALQ VAMP1/1b VEEVVDIMRRodent VAMP2 VDEVVDIMR LDDRADALQ VAMP2-b Rodent VAMP3 VDEVVDIMRLDDRADALQ Bird VAMP1 VEEVVDIMR LDDRADALQ Bird VAMP2 VDEVVDIMR LDNRADALQBird VAMP3 VDEVVDIMR LDDRADALQ Amphibian VAMP2 VDEVVDIMR LDDRADALQAmphibian VAMP3 VDEVVDIMR LDDRADALQ Fish VAMP1 VDEVVDIMR LDDRADALQ FishVAMP2 VDEVVDIMR LDDRADALQ Fish VAMP-3 VDEVVDIMR LDDRADALQ Ray VAMP1VEEVVDIIR LDDRADALQ Sea urchin VAMP VDEVVDIMR LDDRADALQ Insect Syn-A1VDEVVGIMR LGERADQLE Syn-B1 Insect Syn-A2 VDEVVGIMR LGERADQLE Syn-B2Insect Syn-C VDEVVDIMR LDDRADALQ Syn-D Syn-E Segmented worm VAMPVDEVVGMMR LDGRADALQ Cephalopod VAMP VEEVVGIMR LDDRADALQ Gastropod VAMPVDEVVGIMR LDDRAEALQ Round worm SNB1 VDEVVGIMK LDDRADALQ SNB-likeVNEVIDVMR LDHRAEVLQ

Table 7—SNARE motifs of VAMP and Related Proteins. Primate: HumanVAMP-1-1 residues 40-48 and 56-64 of SEQ ID NO: 28; Human VAMP-1-2residues 40-48 and 56-64 of SEQ ID NO: 29; Human VAMP-1-3 residues 40-48and 56-64 of SEQ ID NO: 30; Human VAMP-2 residues 39-47 and 63-71 of SEQID NO: 31; Monkey VAMP-2 residues 39-47 and 63-71 of SEQ ID NO: 32;Human VAMP-3/cellubrevin residues 22-30 and 46-54 of SEQ ID NO: 33;Bovine: Cow VAMP-2 residues 39-47 and 63-71 of SEQ ID NO: 34; Rodent:Rat VAMP-1 residues 40-48 and 56-64 of SEQ ID NO: 35; Rat VAMP-1-bresidues 40-48 and 56-64 of SEQ ID NO: 36; Mouse VAMP-1 residues 40-48and 56-64 of SEQ ID NO: 37; Rat VAMP-2 residues 39-47 and 63-71 of SEQID NO: 38; Rat VAMP-2-b residues 39-47 and 63-71 of SEQ ID NO: 39; MouseVAMP-2 residues 39-47 and 63-71 of SEQ ID NO: 40; Rat VAMP-3/cellubrevinresidues 26-34 and 50-58 of SEQ ID NO: 41; Mouse VAMP-3/cellubrevinresidues 26-34 and 50-58 of SEQ ID NO: 42; Bird: Chicken VAMP-1 residues182-190 and 198-206 of SEQ ID NO: 43; Chicken VAMP-2 residues 37-45 and61-69 of SEQ ID NO: 44; Chicken VAMP-3/cellubrevin residues 26-34 and50-58 of SEQ ID NO: 45; Fish: Zebrafish VAMP-1 residues 41-49 and 57-65of SEQ ID NO: 46; Zebrafish VAMP-2 residues 33-41 and 57-65 of SEQ IDNO: 47; Zebrafish VAMP-3 residues 25-33 and 49-57 of SEQ ID NO: 48; Ray:marbled electric ray VAMP-1 residues 42-50 and 58-66 of SEQ ID NO: 49;Amphibian: Frog VAMP-2 residues 37-45 and 61-69 of SEQ ID NO: 50; FrogVAMP-3 residues 24-32 and 48-56 of SEQ ID NO: 51; Sea urchin VAMPresidues 23-31 and 39-47 of SEQ ID NO: 52; Insect: Fruit fly SynA1residues 31-39 and 47-55 of SEQ ID NO: 53; Fruit fly SynA2 residues54-62 and 70-78 of SEQ ID NO: 54; Fruit fly SynB1 residues 54-62 and70-78 of SEQ ID NO: 55; Fruit fly SynB2 residues 54-62 and 70-78 of SEQID NO: 56; Fruit fly SynC residues 48-56 and 64-72 of SEQ ID NO: 57;Fruit fly SynD residues 67-75 and 83-91 of SEQ ID NO: 58; Fruit fly SynEresidues 67-75 and 83-91 of SEQ ID NO: 59; Segmented worm: Leech VAMPresidues 37-45 and 53-61 of SEQ ID NO: 60; Cephalopod: squid VAMPresidues 47-55 and 63-71 of SEQ ID NO: 61; Gastropod: Pond snail VAMPresidues 40-48 and 56-64 of SEQ ID NO: 62; sea hare VAMP residues 30-38and 46-54 of SEQ ID NO: 63; Round worm: Nematode worm SNB1 residues34-42 and 50-58 of SEQ ID NO: 64; Nematode worm SNB-like residues 40-48and 56-64 of SEQ ID NO: 65.

TABLE 8 SNARE motifs of Syntaxin and Related Proteins Motif OrganismIsoform X1 X2 Primate Syntaxin1A MDEFFEQVE LEDMLESGN Syntaxin1B1MDEFFEQEE LEDMLESGK Syntaxin1B2 MDEFFEQVE LEDMLESGK Primate Syntaxin2-1MDDFFHQVE LEEMLESGK Syntaxin2-2 Syntaxin2-3 Primate Syntaxin3A MDEFFSEIELEEMLESGN Bovine Syntaxin1A MDEFFEQVE LEDMLESGN Syntaxin1B2 LEDMLESGKRodent Syntaxin1A MDEFFEQVE LEDMLESGN Syntaxin1B1 MAEFFEQVE LEDMLESGKSyntaxin1B2 MDEFFEQVE LEDMLESGK Rodent Syntaxin2 MDGFFHQVE LEEMLESGKRodent Syntaxin3A MDEFFSEIE LEEMLESGN Syntaxin3B Rodent Syntaxin3CMDEFFSENF LEEMLESGN Bird Syntaxin1B MDEFFEQVE LEDMLESGK Bird Syntaxin2MDDFFQQVE LEEMLESGN Fish Syntaxin1B MDEFFEQVE LEDMLESGK Fish Syntaxin3MDEFFSQIE LEEMLEGGN Sea urchin Syntaxin1B MEEFFEQVE LEDMLESGN InsectSyntaxin1A MDDFFAQVE LEKMLEEGN Segmented worm Syntaxin1A MEEFFEQVNLEDMLESGN Cephalopod Syntaxin1A MEEFFEQVE LEDMLESGN Gastropod Syntaxin1AMEEFFEQVD LEDMIESGN

Table 8—SNARE motifs of Syntaxin and Related Proteins. Primate: HumanSyntaxin1A residues 30-38 and 165-173 of SEQ ID NO: 66; HumanSyntaxin1B1 residues 29-37 and 164-172 of SEQ ID NO: 67; HumanSyntaxin1B2 residues 29-37 and 164-172 of SEQ ID NO: 68; HumanSyntaxin2-1 residues 29-37 and 168-176 of SEQ ID NO: 69; HumanSyntaxin2-2 residues 29-37 and 168-176 of SEQ ID NO: 70; HumanSyntaxin2-3 residues 29-37 and 168-176 of SEQ ID NO: 71; Human Syntaxin3residues 32-40 and 165-173 of SEQ ID NO: 72; Bovine: Cow Syntaxin1Aresidues 30-38 and 165-173 of SEQ ID NO: 73; Cow Syntaxin1B2 residues29-37 and 164-172 of SEQ ID NO: 74; Rodent: Rat Syntaxin1A residues30-38 and 165-173 of SEQ ID NO: 75; Rat Syntaxin1B2 residues 29-37 and164-172 of SEQ ID NO: 76; Mouse Syntaxin1A residues 30-38 and 165-173 ofSEQ ID NO: 77; Mouse Syntaxin1B1 residues 29-37 and 164-172 of SEQ IDNO: 78; Mouse Syntaxin1B2 residues 29-37 and 164-172 of SEQ ID NO: 79;Rat Syntaxin2 residues 31-39 and 170-178 of SEQ ID NO: 80; MouseSyntaxin2 residues 30-38 and 169-177 of SEQ ID NO: 81; Rat Syntaxin3Aresidues 32-40 and 165-173 of SEQ ID NO: 82; Mouse Syntaxin3A residues32-40 and 165-173 of SEQ ID NO: 83; Mouse Syntaxin3B residues 32-40 and165-173 of SEQ ID NO: 84; Mouse Syntaxin3C residues 32-40 and 147-155 ofSEQ ID NO: 85; Bird: Chicken Syntaxin1B residues 29-37 and 157-165 ofSEQ ID NO: 86; Chicken Syntaxin2 residues 28-36 and 167-175 of SEQ IDNO: 87; Fish: Zebrafish Syntaxin1B residues 29-37 and 164-172 of SEQ IDNO: 88; Zebrafish Syntaxin3 residues 29-37 and 163-171 of SEQ ID NO: 89;sea urchin Syntaxin1B residues 29-37 and 164-172 of SEQ ID NO: 90;Insect: Fruit fly Syntaxin1A residues 33-41 and 168-176 of SEQ ID NO:91; Segmented worm: leech Syntaxin1A residues 36-44 and 171-179 of SEQID NO: 92; Cephalopod: squid Syntaxin1A residues 33-41 and 168-176 ofSEQ ID NO: 93; Gastropod: Pond snail Syntaxin1A residues 32-40 and167-175 of SEQ ID NO: 94; sea hare Syntaxin1A residues 32-40 and 167-175of SEQ ID NO: 95.

As discussed above, the SNARE complex is comprised of the t-SNARESNAP-25 along with another t-SNARE, syntaxin 1 and a v-SNAREVAMP/synaptobrevin. Members of the SNAP-25 family of proteins can bedivided into three structural domains and amino-terminal α-helix ofapproximately 84 residues, an approximately 36 amino acid interhelicalloop and a carboxy-terminal α-helix of approximately 86 residues,depending on the individual member. As will be discussed below, allthree of these regions may be used to target SNAP-25 to the plasmamembrane either alone or in any combination of the three.

The interhelical loop of SNAP-25 appears to be important for conferringtargeting specificity of this SNARE protein to the membrane. Forexample, in one study a membrane-targeting domain comprising residues85-120 of SNAP-25 was shown to localize to the cell membrane SusanaGonzalo et al., SNAP-25 is targeted to the plasma membrane through anovel membrane-binding domain, 274(30) J. Biol. Chem. 21313-21318(1999). This region represents two-thirds of the interhelical loop thatconnects the amino- and carboxy-terminal α-helices of SNAP-25. Thefunction of this targeting domain appears to be independent of SNAREprotein-protein interactions since remove of the SNAP-25 regions thatassociate with either syntaxin or synaptobrevin did not interfere withproper targeting of SNAP-25 to the membrane.

Alignment of SNAP-25 family members revealed two conserved motifspresent within the interhelical loop region responsible for membranetargeting. The first is a cysteine-rich region present at theamino-terminal boundary of the membrane-targeting interhelical loopdomain. One or more of the cysteines present in this motif is fattyacylated via a thioester linkage of palmitate. Palmitoylation of thiscysteine-rich may be important for membrane insertion becauseelimination of these cysteine residues results in a loss of SNAP-25membrane-targeting.

The second is a five-amino acid motif located at the carboxy-terminalboundary of the membrane-targeting interhelical loop domain QPXRV (SEQID NO: 135) or QPXRI (SEQ ID NO: 136). This motif is believed to play arole in membrane association, see, e.g., Gonzalo et al., supra, (1999);Philip Washbourne et al., Cysteine residues of SNAP-25 are required forSNARE disassembly and exocytosis, but not for membrane targeting, 357(Pt3) Biochem. J. 625-634 (2001).

The α-helices of the various SNARE complex members seems to be involvedin protein-protein interactions between members. For example, solutionof the crystal structure of the SNARE complex reveals that SNAP-25,syntaxin and synaptobrevin appear to favor a heterotrimeric, parallelfour-helix bundle association, see, e.g., R. Bryan Sutton et al.,Crystal structure of a SNARE complex involved in synaptic exocytosis at2.4 Å resolution, 395(6700) Nature 347-353 (1998). This analysisindicated an extensive intertwining of the α-helices with theamino-terminal region of the bundle comprising interactions between theamino-terminal α-helix of SNAP-25 with syntaxin, several centralassociations amongst all three members and an association betweensyntaxin and synaptobrevin at the carboxyl-terminal portion of thefour-helix bundle.

Protein-protein interactions between the α-helices of SNARE complexmembers appear to be another way of localizing SNAP-25 to the membrane.For example, co-expression of SNAP-25 with syntaxin results in targetingSNAP-25 to the membrane in the absence of a functional interhelical loopsuggesting that protein-protein interactions between these two t-SNAREscan target Clostridial toxin substrates to the membrane, see, e.g.,Washbourne et al., supra, (2001).

Members of the Syntaxin family of proteins can be divided into severalstructural domains. In the amino-terminal half of the protein containsan Habc region comprising three α-helix domains located at amino acids30-60, 69-104 and 110-154. The carboxy-terminal half of Syntaxin-1contains an α-helix of approximately 52-69 residues, depending on theindividual member and an approximately 23 amino acid membrane anchoringdomain. As will be discussed below, regions comprising the membraneanchoring domain of Syntaxin may be used to target Clostridial toxinsubstrates to the plasma membrane.

The Clostridial toxin substrates disclosed in the present specificationinclude, in part, a membrane targeting domain. As used herein, the term“membrane targeting domain” is synonymous with “MTD” and means a SNAP-25or Syntaxin peptide which directs a Clostridial toxin substrate to thecell membrane. Any and all SNAP-25 or Syntaxin membrane targetingdomains can be used in aspects of the present invention, with theproviso that the Clostridial toxin substrate maintains the property tobe cleaved by a Clostridial toxin. Non-limiting examples include,without limitation, naturally occurring membrane targeting domainspresent in SNAP-25, naturally occurring SNAP-25 MTD variants, andnon-naturally occurring SNAP-25 MTD variants, such as, e.g., geneticallyengineered SNAP-25 MTD variants, produced, e.g., by random mutagenesisor rational designed and SNAP-25 MTD peptidomimetics; and naturallyoccurring membrane targeting domains present in Syntaxin, naturallyoccurring Syntaxin MTD variants, and non-naturally occurring SyntaxinMTD variants, such as, e.g., genetically engineered Syntaxin MTDvariants, produced, e.g., by random mutagenesis or rational designed andSyntaxin MTD peptidomimetics.

Thus, in an embodiment a Clostridial toxin substrate comprises, in part,the membrane targeting domain comprising a region from SNAP-25sufficient to target a toxin substrate disclosed in the presentspecification to the membrane. In an aspect of this embodiment, themembrane targeting domain comprising a region from the interhelicalregion of SNAP-25 sufficient to target a toxin substrate disclosed inthe present specification to the membrane. In an aspect of thisembodiment the membrane targeting domain comprises the amino acids85-120 of SEQ ID NO: 1. It is envisioned that an interhelical loopregion from SNAP-25 of any and all lengths can comprise the membranetargeting domain with the proviso that the loop region is sufficient totarget a toxin substrate disclosed in the present specification to themembrane. Thus, aspects of this embodiment may include an interhelicalloop region comprising, e.g., at least 35 residues from amino acids85-120 of SEQ ID NO: 1, at least 30 residues from amino acids 85-120 ofSEQ ID NO: 1, at least 25 residues from amino acids 85-120 of SEQ ID NO:1, at least 20 residues from amino acids 85-120 of SEQ ID NO: 1, atleast 15 residues from amino acids 85-120 of SEQ ID NO: 1, at least 10residues from amino acids 85-120 of SEQ ID NO: 1 or at least 5 residuesfrom amino acids 85-120 of SEQ ID NO: 1. Further aspects of thisembodiment may include an interhelical loop region comprising, e.g., atmost 35 residues from amino acids 85-120 of SEQ ID NO: 1, at most 30residues from amino acids 85-120 of SEQ ID NO: 1, at most 25 residuesfrom amino acids 85-120 of SEQ ID NO: 1, at most 20 residues from aminoacids 85-120 of SEQ ID NO: 1, at most 15 residues from amino acids85-120 of SEQ ID NO: 1, at most 10 residues from amino acids 85-120 ofSEQ ID NO: 1 or at most 5 residues from amino acids 85-120 of SEQ ID NO:1.

In another aspect of this embodiment a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprises amino acidsCGLCVCPCNK (SEQ ID NO: 128). In another aspect of this embodiment themembrane targeting domain comprises amino acids CGLFICPCNK (SEQ ID NO:129). In another aspect of this embodiment the membrane targeting domaincomprises amino acids CGLCSCPCNK (SEQ ID NO: 130). In another aspect ofthis embodiment the membrane targeting domain comprises amino acidsCGLCPCPCNK (SEQ ID NO: 131). In another aspect of this embodiment themembrane targeting domain comprises amino acids CGICVCPWKK (SEQ ID NO:132). In another aspect of this embodiment the membrane targeting domaincomprises amino acids CGICVLPCNK (SEQ ID NO: 133). In another aspect ofthis embodiment the membrane targeting domain comprises amino acidsCGLCVLPWNK (SEQ ID NO: 134).

In another embodiment a Clostridial toxin substrate comprises, in part,the membrane targeting domain comprises the amino acids QPXRV (SEQ IDNO: 135), where X is any amino acid. In another aspect of thisembodiment the membrane targeting domain comprises amino acids QPXR1(SEQ ID NO: 136), where X is any amino acid. In another aspect of thisembodiment the membrane targeting domain comprises amino acids QPARV(SEQ ID NO: 137). In another aspect of this embodiment the membranetargeting domain comprises amino acids QPQRV (SEQ ID NO: 138). Inanother aspect of this embodiment the membrane targeting domaincomprises amino acids QPGRV (SEQ ID NO: 139). In another aspect of thisembodiment the membrane targeting domain comprises amino acids QPSR1(SEQ ID NO: 140). In another aspect of this embodiment the membranetargeting domain comprises amino acids QPMRM (SEQ ID NO: 141). Inanother aspect of this embodiment the membrane targeting domaincomprises amino acids QPR1 (SEQ ID NO: 142).

In another embodiment a Clostridial toxin substrate comprises, in part,the membrane targeting domain comprises amino acids from theamino-terminal α-helix of SNAP-25 sufficient to target a toxin substratedisclosed in the present specification to the membrane. In an aspect ofthis embodiment the membrane targeting domain comprises the amino acids1-84 of SEQ ID NO: 1. It is envisioned that an amino-terminal α-helixfrom SNAP-25 of any and all lengths can comprise the membrane targetingdomain with the proviso that the loop region is sufficient to target atoxin substrate disclosed in the present specification to the membrane.Thus, aspects of this embodiment may include an amino-terminal α-helixregion comprising, e.g., at least 80 residues from amino acids 1-84 ofSEQ ID NO: 1, at least 75 residues from amino acids 1-84 of SEQ ID NO:1, at least 70 residues from amino acids 1-84 of SEQ ID NO: 1, at least65 residues from amino acids 1-84 of SEQ ID NO: 1, at least 60 residuesfrom amino acids 1-84 of SEQ ID NO: 1, at least 55 residues from aminoacids 1-84 of SEQ ID NO: 1, at least 50 residues from amino acids 1-84of SEQ ID NO: 1, at least 45 residues from amino acids 1-84 of SEQ IDNO: 1, at least 40 residues from amino acids 1-84 of SEQ ID NO: 1, atleast 35 residues from amino acids 1-84 of SEQ ID NO: 1, at least 30residues from amino acids 1-84 of SEQ ID NO: 1, at least 25 residuesfrom amino acids 1-84 of SEQ ID NO: 1, at least 20 residues from aminoacids 1-84 of SEQ ID NO: 1, at least 15 residues from amino acids 1-84of SEQ ID NO: 1, at least 10 residues from amino acids 1-84 of SEQ IDNO: 1 or at least 5 residues from amino acids 1-84 of SEQ ID NO: 1.Further aspects of this embodiment may include an amino-terminal α-helixregion comprising, e.g., at most 80 residues from amino acids 1-84 ofSEQ ID NO: 1, at most 75 residues from amino acids 1-84 of SEQ ID NO: 1,at most 70 residues from amino acids 1-84 of SEQ ID NO: 1, at most 65residues from amino acids 1-84 of SEQ ID NO: 1, at most 60 residues fromamino acids 1-84 of SEQ ID NO: 1, at most 55 residues from amino acids1-84 of SEQ ID NO: 1, at most 50 residues from amino acids 1-84 of SEQID NO: 1, at most 45 residues from amino acids 1-84 of SEQ ID NO: 1, atmost 40 residues from amino acids 1-84 of SEQ ID NO: 1, at most 35residues from amino acids 1-84 of SEQ ID NO: 1, at most 30 residues fromamino acids 1-84 of SEQ ID NO: 1, at most 25 residues from amino acids1-84 of SEQ ID NO: 1, at most 20 residues from amino acids 1-84 of SEQID NO: 1, at most 15 residues from amino acids 1-84 of SEQ ID NO: 1, atmost 10 residues from amino acids 1-84 of SEQ ID NO: 1 or at most 5residues from amino acids 1-84 of SEQ ID NO: 1.

In yet another embodiment a Clostridial toxin substrate comprises, inpart, the membrane targeting domain comprises amino acids from thecarboxy-terminal α-helix of SNAP-25 sufficient to target a toxinsubstrate disclosed in the present specification to the membrane. In anaspect of this embodiment the membrane targeting domain comprises theamino acids 121-206 of SEQ ID NO: 1. It is envisioned that ancarboxy-terminal α-helix from SNAP-25 of any and all lengths cancomprise the membrane targeting domain with the proviso that the loopregion is sufficient to target a toxin substrate disclosed in thepresent specification to the membrane. Thus, aspects of this embodimentmay include an carboxy-terminal α-helix region comprising, e.g., atleast 80 residues from amino acids 121-206 of SEQ ID NO: 1; at least 75residues from amino acids 121-206 of SEQ ID NO: 1, at least 70 residuesfrom amino acids 121-206 of SEQ ID NO: 1, at least 65 residues fromamino acids 121-206 of SEQ ID NO: 1, at least 60 residues from aminoacids 121-206 of SEQ ID NO: 1, at least 55 residues from amino acids121-206 of SEQ ID NO: 1, at least 50 residues from amino acids 121-206of SEQ ID NO: 1, at least 45 residues from amino acids 121-206 of SEQ IDNO: 1, at least 40 residues from amino acids 121-206 of SEQ ID NO: 1, atleast 35 residues from amino acids 121-206 of SEQ ID NO: 1, at least 30residues from amino acids 121-206 of SEQ ID NO: 1, at least 25 residuesfrom amino acids 121-206 of SEQ ID NO: 1, at least 20 residues fromamino acids 121-206 of SEQ ID NO: 1, at least 15 residues from aminoacids 121-206 of SEQ ID NO: 1, at least 10 residues from amino acids121-206 of SEQ ID NO: 1 or at least 5 residues from amino acids 121-206of SEQ ID NO: 1. Further aspects of this embodiment may include ancarboxy-terminal α-helix region comprising, e.g., at most 85 residuesfrom amino acids 121-206 of SEQ ID NO: 1; at most 80 residues from aminoacids 121-206 of SEQ ID NO: 1; at most 75 residues from amino acids121-206 of SEQ ID NO: 1, at most 70 residues from amino acids 121-206 ofSEQ ID NO: 1, at most 65 residues from amino acids 121-206 of SEQ ID NO:1, at most 60 residues from amino acids 121-206 of SEQ ID NO: 1, at most55 residues from amino acids 121-206 of SEQ ID NO: 1, at most 50residues from amino acids 121-206 of SEQ ID NO: 1, at most 45 residuesfrom amino acids 121-206 of SEQ ID NO: 1, at most 40 residues from aminoacids 121-206 of SEQ ID NO: 1, at most 35 residues from amino acids121-206 of SEQ ID NO: 1, at most 30 residues from amino acids 121-206 ofSEQ ID NO: 1, at most 25 residues from amino acids 121-206 of SEQ ID NO:1, at most 20 residues from amino acids 121-206 of SEQ ID NO: 1, at most15 residues from amino acids 121-206 of SEQ ID NO: 1, at most 10residues from amino acids 121-206 of SEQ ID NO: 1 or at most 5 residuesfrom amino acids 121-206 of SEQ ID NO: 1.

In another embodiment a Clostridial toxin substrate comprises, in part,the membrane targeting domain comprising a region from Syntaxinsufficient to target a toxin substrate disclosed in the presentspecification to the membrane. In an aspect of this embodiment, themembrane targeting domain comprising a region from the membraneanchoring domain of Syntaxin sufficient to target a toxin substratedisclosed in the present specification to the membrane. In an aspect ofthis embodiment the membrane targeting domain comprises the amino acids266-288 of SEQ ID NO: 66. It is envisioned that an membrane anchoringdomain from Syntaxin of any and all lengths can comprise the membranetargeting domain with the proviso that the loop region is sufficient totarget a toxin substrate disclosed in the present specification to themembrane. Thus, aspects of this embodiment may include an interhelicalloop region comprising, e.g., at least 20 residues from amino acids266-288 of SEQ ID NO: 66; at least 15 residues from amino acids 266-288of SEQ ID NO: 66, Or at least 10 residues from amino acids 266-288 ofSEQ ID NO: 66. Further aspects of this embodiment may include anmembrane anchoring domain comprising, e.g., at most 20 residues fromamino acids 266-288 of SEQ ID NO: 66; at most 15 residues from aminoacids 266-288 of SEQ ID NO: 66 or at most 10 residues from amino acids266-288 of SEQ ID NO: 66.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of human Syntaxin-1Aof SEQ ID NO: 66. In another aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domaincomprising amino acids IMIIICCVILGIVIASTVGGIFA, which corresponds toresidues 266-288 of SEQ ID NO: 66. In another aspect of this embodiment,a Clostridial toxin substrate comprises, in part, the membrane targetingdomain of human Syntaxin-1B1 of SEQ ID NO: 67. In another aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain comprising amino acids IIIIICCWLGWLASSIGCTLGL,which corresponds to residues 265-288 of SEQ ID NO: 67. In anotheraspect of this embodiment, a Clostridial toxin substrate comprises, inpart, the membrane targeting domain of human Syntaxin-1B2 of SEQ ID NO:68. In another aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsIMIIICCWLGWLASSIGGTLGL, which corresponds to residues 265-288 of SEQ IDNO: 67. In another aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain of humanSyntaxin-2-1 of SEQ ID NO: 69. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids LMFIIICVIVLLVILGIILATTLS, whichcorresponds to residues 264-287 of SEQ ID NO: 69. In another aspect ofthis embodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain of human Syntaxin-2-2 of SEQ ID NO: 70. Inanother aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsWIIIAVSWLVVIIVLIIGLSVGK, which corresponds to residues 264-288 of SEQ IDNO: 70. In another aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain of humanSyntaxin-2-3 of SEQ ID NO: 71. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids WIIIAVSWLVAIIALIIGLSVGK, which correspondsto residues 264-288 of SEQ ID NO: 71. In another aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain of human Syntaxin-3 of SEQ ID NO: 72. Inanother aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsLIIIIVLWVLLGILALIIGISVGLN, which corresponds to residues 264-289 of SEQID NO: 72.

In another aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of cow Syntaxin-1A ofSEQ ID NO: 73. In another aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain comprisingamino acids IMIVICCVVLGIVIASTFGGIFG, which corresponds to residues266-288 of SEQ ID NO: 67.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of rat Syntaxin-1A ofSEQ ID NO: 75. In another aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain comprisingamino acids IMIIICCVILGIIIASTIGGIFG, which corresponds to residues266-288 of SEQ ID NO: 75. In an aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domain of ratSyntaxin-1B2 of SEQ ID NO: 76. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids IMIIICCWLGWLASSIGGTLGL, which correspondsto residues 265-288 of SEQ ID NO: 76. In an aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain of rat Syntaxin-2 of SEQ ID NO: 80. In another aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain comprising amino acids WIIAVWAVIAVLALIIGLSVGK,which corresponds to residues 267-290 of SEQ ID NO: 80. In an aspect ofthis embodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain of mouse Syntaxin-2 of SEQ ID NO: 81. Inanother aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsWIIAAVAVAVIAVLALIIGLSVGK, which corresponds to residues 266-289 of SEQID NO: 81. In an aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain of ratSyntaxin-3A of SEQ ID NO: 82. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids LIIIIVIVWLLGILALIIGISVGLK, whichcorresponds to residues 264-289 of SEQ ID NO: 82. In an aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain of mouse Syntaxin-3A of SEQ ID NO: 83. Inanother aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsLIIIIVVVVVLLGILALIIGLSVGLK, which corresponds to residues 264-289 of SEQID NO: 83. In an aspect of this embodiment, a Clostridial toxinsubstrate comprises, in part, the membrane targeting domain of mouseSyntaxin-3B of SEQ ID NO: 84. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids IMIMICClILAIILASTIG, which corresponds toresidues 265-283 of SEQ ID NO: 84. In an aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain of mouse Syntaxin-3C of SEQ ID NO: 85. In another aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain comprising amino acidsIMIMICClILAIILASTIGGIFA, which corresponds to residues 247-269 of SEQ IDNO: 85.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of chicken Syntaxin-1Aof SEQ ID NO: 86. In another aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domaincomprising amino acids IMIIIFWVLGWLSPVICGTLGL, which corresponds toresidues 259-282 of SEQ ID NO: 86. In an aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain of chicken Syntaxin-2 of SEQ ID NO: 87. In another aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain comprising amino acidsWIIIIVSLVLIAVIGIIIGLSVGIR, which corresponds to residues 263-288 of SEQID NO: 87.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of zebrafishSyntaxin-1A of SEQ ID NO: 88. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids IMIIICCVILGVVLRSSIGGTLGF, whichcorresponds to residues 265-288 of SEQ ID NO: 88. In an aspect of thisembodiment, a Clostridial toxin substrate comprises, in part, themembrane targeting domain of zebrafish Syntaxin-3 of SEQ ID NO: 89. Inanother aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain comprising amino acidsIIIIVSWLVILAIIALIVGISVGLKR, which corresponds to residues 262-288 of SEQID NO: 89.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of sea urchinSyntaxin-1A of SEQ ID NO: 90. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids YIAICCGVALGILILVLIIVLA, which correspondsto residues 264-286 of SEQ ID NO: 90.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of fruit flySyntaxin-1A of SEQ ID NO: 91. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids IMILICLTVLGILAASYVSSYFM, which correspondsto residues 269-291 of SEQ ID NO: 91.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of leech Syntaxin-1Aof SEQ ID NO: 92. In another aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domaincomprising amino acids IIILICVSVLILIVGGSLLGIFIP, which corresponds toresidues 272-295 of SEQ ID NO: 92.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of squid Syntaxin-1Aof SEQ ID NO: 93. In another aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domaincomprising amino acids IAILVCLVILVLVIVSTVGGVFGG, which corresponds toresidues 269-292 of SEQ ID NO: 93.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of snail Syntaxin-1Aof SEQ ID NO: 94. In another aspect of this embodiment, a Clostridialtoxin substrate comprises, in part, the membrane targeting domaincomprising amino acids IMIIICVCVLIIILVGILGGTFG, which corresponds toresidues 268-290 of SEQ ID NO: 94.

In an aspect of this embodiment, a Clostridial toxin substratecomprises, in part, the membrane targeting domain of sea hareSyntaxin-1A of SEQ ID NO: 95. In another aspect of this embodiment, aClostridial toxin substrate comprises, in part, the membrane targetingdomain comprising amino acids IMILVCLAILIIILVGVIGGTLG, which correspondsto residues 268-290 of SEQ ID NO: 95.

Clostridial toxin substrates disclosed in the present specificationinclude, in part, a fluorescent member. As used herein, the term“fluorescent member” means any peptide that can either inherentlyabsorbs light energy of a certain wavelength and emits light energy of adifferent wavelength or is part of a complex that absorbs light energyof a certain wavelength and emits light energy of a differentwavelength. Non-limiting examples of a fluorescent member includefluorescent proteins and fluorophore binding proteins.

Clostridial toxin substrates disclosed in the present specificationinclude, in part, a fluorescent protein. As used herein, the term“fluorescent protein” means a peptide which absorbs light energy of acertain wavelength and emits light energy of a different wavelength andencompass those which emit in a variety of spectra, including violet,blue, cyan, green, yellow, orange and red, see Table 9. It is envisionedthat fluorescent proteins derived from any of a variety of species canbe useful in aspects of the present invention including, but not limitedto, Aequorea fluorescent proteins, Anemonia fluorescent proteins,Anthozoa fluorescent proteins, Discosoma fluorescent proteins, Entacmeaefluorescent proteins, Heteractis fluorescent proteins, Montastreafluorescent proteins, Renilla fluorescent proteins, Zoanthus fluorescentproteins, and fluorescent proteins from other organisms. Fluorescentproteins useful in the invention encompass, without limitation, wildtype fluorescent proteins, naturally occurring variants, and geneticallyengineered variants, produced, e.g., by random mutagenesis or rationaldesigned, and active peptide fragments derived from an organism.Fluorescent proteins useful in aspects of the invention include, e.g.,those which have been genetically engineered for superior performancesuch as, without limitation, altered excitation or emission wavelengths;enhanced brightness, pH resistance, stability or speed of fluorescentprotein formation; photoactivation; or reduced oligomerization orphotobleaching, see, e.g., Brendan P. Cormack et al., FACS-optimizedMutants of the Green Fluorescent Protein (GFP), U.S. Pat. No. 5,804,387(Sep. 8, 1998); Roger Y. Tsien & Roger Heim, Modified Green FluorescentProteins, U.S. Pat. No. 6,800,733 (Oct. 5, 2004); Roger Y. Tsien et al.,Long Wavelength Engineered Fluorescent Proteins, U.S. Pat. No. 6,780,975(Aug. 24, 2004); and Roger Y. Tsien et al., Fluorescent Protein SensorsFor Measuring the pH of a Biological Sample, U.S. Pat. No. 6,627,449(Sep. 30, 2003). It is understood that a fluorescent protein can beengineered for improved protein expression by converting wild typecodons to other codons more efficiently utilized in the cells whichserve to express the Clostridial toxin substrate, see, e.g., Brian Seed& Jurgen Haas, High Level Expression of Proteins, U.S. Pat. No.5,795,737 (Aug. 18, 1998).

It is also envisioned that any of a variety of active protein fragmentscan be useful in aspects of the present invention with the proviso thatthese active fragments retain the ability to emit light energy in arange suitable for the proper operation of aspects of the presentinvention, such as, e.g. 420-460 nm for blue emitting fluorescentproteins, 460-500 nm for cyan emitting fluorescent proteins, 500-520 nmfor green emitting fluorescent proteins, 520-550 nm for yellow emittingfluorescent proteins and for 550-740 nm for red emitting fluorescentproteins. Thus, aspects of this embodiment can include active fragmentsof fluorescent proteins that retain the ability to emit light energy ina range suitable for the proper operation of aspects of the presentinvention having a length of, e.g., at least 50 amino acids, at least 60amino acids, at least 70 amino acids, at least 80 amino acids, at least90 amino acids, at least 100 amino acids, at least 125 amino acids, atleast 150 amino acids, at least 175 amino acids and at least 200 aminoacids. Other aspects of this embodiment, can include active fragments offluorescent proteins that retain the ability to emit light energy in arange suitable for the proper operation of aspects of the presentinvention having a length of, e.g., at most 50 amino acids, at most 60amino acids, at most 70 amino acids, at most 80 amino acids, at most 90amino acids, at most 100 amino acids, at most 125 amino acids, at most150 amino acids, at most 175 amino acids and at most 200 amino acids.

Non-limiting examples of fluorescent proteins that may beoperably-linked to a CoNT substrate disclosed in the specificationinclude, e.g., photoproteins, such as, e.g., aequorin; obelin; Aequoreafluorescent proteins, such, e.g., green fluorescent proteins (GFP, EGFP,AcGFP1), cyan fluorescent proteins (CFP, ECFP), blue fluorescentproteins (BFP, EBFP), red fluorescent proteins (RFP), yellow fluorescentproteins (YFP, EYFP), ultraviolet fluorescent protein (GFPuv), theirfluorescence-enhancement variants, their peptide destabilizationvariants, and the like; coral reef fluorescent proteins, such, e.g.,Discosoma red fluorescent proteins (DsRed, DsRed1, DsRed2, andDsRed-Express), Anemonia red fluorescent proteins (AsRed and AsRed2),Heteractis far-red fluorescent proteins (HcRed, HcRed1), Anemonia cyanfluorescent proteins (AmCyan, AmCyan1), Zoanthus green fluorescentproteins (ZsGreen, ZsGreen1), Zoanthus yellow fluorescent proteins(ZsYellow, ZsYellow1), their fluorescence-enhancement variants, theirpeptide destabilization variants, and the like; Renilla reniformis greenfluorescent protein (Vitality hrGFP), its fluorescence-enhancementvariants, its peptide destabilization variants, and the like; and GreatStar Coral fluorescent proteins, such, e.g., Montastrea cavernosafluorescent protein (Monster Green® Fluorescent Protein), itsfluorescence-enhancement variants, its peptide destabilization variants,and the like. One skilled in the art understands that these and avariety of other fluorescent proteins can be useful as a fluorescentprotein in aspects of the invention, see, e.g., JenniferLippincott-Schwartz & George H. Patterson, Development and Use ofFluorescent Protein Markers in Living Cells, 300(5616) Science 87-91(2003); and Jin Zhang et al., 3(12) Nat. Rev. Mol. Cell Biol. 906-918(2002). One skilled in the art understands that these and many otherfluorescent proteins, including species orthologs and paralogs of theabove described naturally occurring fluorescent proteins as well asengineered fluorescent proteins can be useful as a fluorescent proteindisclosed in aspects of the present specification. CoNT substratesdisclosed in the present specification containing, in part, suchfluorescent proteins can be prepared and expressed using standardmethods see, e.g., Living Colorse User Manual PT2040-1 (PRI1Y691), BDBiosciences-Clontech, (Nov. 26 2001); BD Living Colors™ User ManualVolume II: Reef Coral Fluorescent Proteins, PT3404-1 (PR37085), BDBiosciences-Clontech, (Jul. 17, 2003); Monster Green Florescent ProteinpHMCFP Vector, TB320, Promega Corp., (May, 2004); and Vitality hrGFPMammalian Expression Vectors, Instruction Manual (rev. 064007g),Stratagene, Inc. Expression vectors suitable for bacterial, mammalianand other expression of fluorescent proteins are available from avariety of commercial sources including BD Biosciences Clontech (PaloAlto, Calif.); Promega Corp. (Madison, Wis.) and Stratagene, Inc. (LaJolla, Calif.).

In an embodiment, the fluorescent protein is a green fluorescentprotein. As used herein, the term “green fluorescent protein” issynonymous with “GFP” and means a protein which absorbs light of acertain wavelength and emits peak light energy of wavelengths in therange of 500-520 nm. Green fluorescent proteins useful in the inventioninclude, without limitation, the AcGFP1 of SEQ ID NO: 143, geneticallyengineered AcGFP1 variants and active AcGFP1 fragments thereof thatretain the ability to emit peak light energy in the range of 500-520 nm,the ZsGreen of SEQ ID NO: 144, genetically engineered ZsGreen variantsand active ZsGreen fragments thereof that retain the ability to emitpeak light energy in the range of 500-520 nm, the EGFP of SEQ ID NO:145, genetically engineered ECFP variants and active ECFP fragmentsthereof that retain the ability to emit peak light energy in the rangeof 500-520 nm, the Monster Green Fluorescent Protein (MGFP) of SEQ IDNO: 146, genetically engineered MGFP variants and active MGFP fragmentsthereof that retain the ability to emit peak light energy in the rangeof 500-520 nm, the Vitalitye hrGFP of SEQ ID NO: 147, geneticallyengineered hrGFP variants and active hrGFP fragments thereof that retainthe ability to emit peak light energy in the range of 500-520 nm, aswell as, naturally-occurring GFPs, naturally occurring GFP variants,genetically engineered GFP variants and active GFP fragments thereofthat retain the ability to emit peak light energy in the range of500-520 nm. As non-limiting examples, Renilla-derived fluorescentproteins such as, e.g., the dimeric Renilla mulleri GFP, which hasnarrow excitation (498 nm) and emission (509 nm) peaks, see, e.g., BeauPeelle et al., Characterization and use of green fluorescent proteinsfrom Renilla mulleri and Ptilosarcus guernyi for the human cell displayof functional peptides, 20(6) J. Protein Chem. 507-519 (2001); andAequorea-derived fluorescent proteins as described in, e.g., Roger Y.Tsien & Roger Heim, Modified Green Fluorescent Proteins, U.S. Pat. No.5,625,048 (Apr. 29, 1997), U.S. Pat. No. 6,319,669 (Nov. 20, 2001),6,066,476 (May 23, 2000) and U.S. Pat. No. 6,800,733 (Oct. 5, 2004).

Thus, in aspects of this embodiment, the fluorescent protein can be aGFP that emits peak s light in the range of 500-520 nm which has, e.g.,at least 70% amino acid identity with the AcGFP1 of SEQ ID NO: 143, atleast 75% amino acid identity with the AcGFP1 of SEQ ID NO: 143, atleast 80% amino acid identity with the AcGFP1 of SEQ ID NO: 143, atleast 85% amino acid identity with the AcGFP1 of SEQ ID NO: 143, atleast 90% amino acid identity with the AcGFP1 of SEQ ID NO: 143 or atleast 95% amino acid identity with the AcGFP1 of SEQ ID NO: 143. Inother aspects of this embodiment, the fluorescent protein is a GFP thatemits light in the range of 500-520 nm which has, e.g., at most one,two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the AcGFP1 of SEQ ID NO: 143.

In other aspects of this embodiment, the fluorescent protein can be aGFP that emits light in the range of 500-520 nm which has, e.g., atleast 70% amino acid identity with the ZsGreen of SEQ ID NO: 144, atleast 75% amino acid identity with the ZsGreen of SEQ ID NO: 144, atleast 80% amino acid identity with the ZsGreen of SEQ ID NO: 144, atleast 85% amino acid identity with the ZsGreen of SEQ ID NO: 144, atleast 90% amino acid identity with the ZsGreen of SEQ ID NO: 144 or atleast 95% amino acid identity with the ZsGreen of SEQ ID NO: 144. Instill other aspects of this embodiment, the fluorescent protein is a GFPthat emits light in the range of 500-520 nm which has, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the ZsGreen of SEQ ID NO: 144.

In other aspects of this embodiment, the fluorescent protein can be aGFP that emits light in the range of 500-520 nm which has, e.g., atleast 70% amino acid identity with the EGFP of SEQ ID NO: 145, at least75% amino acid identity with the EGFP of SEQ ID NO: 145, at least 80%amino acid identity with the EGFP of SEQ ID NO: 145, at least 85% aminoacid identity with the EGFP of SEQ ID NO: 145, at least 90% amino acididentity with the EGFP of SEQ ID NO: 145 or at least 95% amino acididentity with the EGFP of SEQ ID NO: 145. In still other aspects of thisembodiment, the fluorescent protein is a GFP that emits light in therange of 500-520 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the EGFP of SEQ ID NO: 145.

In other aspects of this embodiment, the fluorescent protein can be aGFP that emits light in the range of 500-520 nm which has, e.g., atleast 70% amino acid identity with the MGFP of SEQ ID NO: 146, at least75% amino acid identity with the MGFP of SEQ ID NO: 146, at least 80%amino acid identity with the MGFP of SEQ ID NO: 146, at least 85% aminoacid identity with the MGFP of SEQ ID NO: 146, at least 90% amino acididentity with the MGFP of SEQ ID NO: 146 or at least 95% amino acididentity with the MGFP of SEQ ID NO: 146. In still other aspects of thisembodiment, the fluorescent protein is a GFP that emits light in therange of 500-520 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the MGFP of SEQ ID NO: 146.

In other aspects of this embodiment, the fluorescent protein can be aGFP that emits light in the range of 500-520 nm which has, e.g., atleast 70% amino acid identity with the hrGFP of SEQ ID NO: 147, at least75% amino acid identity with the hrGFP of SEQ ID NO: 147, at least 80%amino acid identity with the hrGFP of SEQ ID NO: 147, at least 85% aminoacid identity with the hrGFP of SEQ ID NO: 147, at least 90% amino acididentity with the hrGFP of SEQ ID NO: 147 or at least 95% amino acididentity with the hrGFP of SEQ ID NO: 147. In still other aspects ofthis embodiment, the fluorescent protein is a GFP that emits light inthe range of 500-520 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the hrGFP of SEQ ID NO: 147.

In another embodiment, the fluorescent protein is a cyan fluorescentprotein. As used herein, the term “cyan fluorescent protein” issynonymous with “CFP” and means a protein which absorbs light of acertain wavelength and emit peak light energy of wavelengths in therange of 460-500 nm. Cyan fluorescent proteins useful in the inventioninclude, without limitation, the ECFP of SEQ ID NO: 148, geneticallyengineered ECFP variants and active ECFP fragments thereof that retainthe ability to emit peak light energy in the range of 460-500 nm, theAmCyan of SEQ ID NO: 149, genetically engineered AmCyan variants andactive AmCyan fragments thereof that retain the ability to emit peaklight energy in the range of 460-500 nm, as well as, naturally-occurringcyan fluorescent proteins, naturally occurring CFP variants, geneticallyengineered CFP variants and active CFP fragments thereof that retain theability to emit peak light energy in the range of 460-500 nm. As anon-limiting example, the CFP variant known as “CGFP” contains aThr203Tyr substitution that changes the excitation and emissionwavelengths of the ECFP of SEQ ID NO: 148 to a range between CFP andEGFP; and Aequorea-derived fluorescent proteins as described in, e.g.,Roger Y. Tsien & Roger Heim, Modified Green Fluorescent Proteins, U.S.Pat. No. 5,625,048 (Apr. 29, 1997), U.S. Pat. No. 6,319,669 (Nov. 20,2001), U.S. Pat. No. 6,066,476 (May 23, 2000) and U.S. Pat. No.6,800,733 (Oct. 5, 2004).

Thus, in aspects of this embodiment, the fluorescent protein is a CFPthat emits light in the range of 460-500 nm which has, e.g., at least70% amino acid identity with the ECFP of SEQ ID NO: 148, at least 75%amino acid identity with the ECFP of SEQ ID NO: 148, at least 80% aminoacid identity with the ECFP of SEQ ID NO: 148, at least 85% amino acididentity with the ECFP of SEQ ID NO: 148, at least 90% amino acididentity with the ECFP of SEQ ID NO: 148 or at least 95% amino acididentity with the ECFP of SEQ ID NO: 148. In other aspects of thisembodiment, the fluorescent protein is a CFP that emits light in therange of 460-500 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the ECFP of SEQ ID NO: 148.

In other aspects of this embodiment, the fluorescent protein is a CFPthat emits light in the range of 460-500 nm which has, e.g., at least70% amino acid identity with the AmCyan of SEQ ID NO: 149, at least 75%amino acid identity with the AmCyan of SEQ ID NO: 149, at least 80%amino acid identity with the AmCyan of SEQ ID NO: 149, at least 85%amino acid identity with the AmCyan of SEQ ID NO: 149, at least 90%amino acid identity with the AmCyan of SEQ ID NO: 149 or at least 95%amino acid identity with the AmCyan of SEQ ID NO: 149. In still otheraspects of this embodiment, the fluorescent protein is a CFP that emitslight in the range of 460-500 nm which has, e.g., at most one, two,three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the AmCyan of SEQ ID NO: 149.

In yet another embodiment, the fluorescent protein is a blue fluorescentprotein. As used herein, the term “blue fluorescent protein” issynonymous with “BFP” and means a protein which absorbs light of acertain wavelength and emit peak light energy of wavelengths in therange of 420-460 nm. Blue fluorescent proteins useful in the inventioninclude, without limitation, the EBFP of SEQ ID NO: 150, geneticallyengineered EBFP variants and active EBFP fragments thereof that retainthe ability to emit peak light energy in the range of 420-460 nm, aswell as, naturally-occurring blue fluorescent proteins, naturallyoccurring BFP variants, genetically engineered BFP variants and activeBFP fragments thereof that retain the ability to emit peak light energyin the range of 420-460 nm. As non-limiting examples, seeAequorea-derived fluorescent proteins as described in, e.g., Roger Y.Tsien & Roger Heim, Modified Green Fluorescent Proteins, U.S. Pat. No.5,625,048 (Apr. 29, 1997), U.S. Pat. No. 6,319,669 (Nov. 20, 2001), U.S.Pat. No. 6,066,476 (May 23, 2000) and U.S. Pat. No. 6,800,733 (Oct. 5,2004).

Thus, in aspects of this embodiment, the fluorescent protein is a BFPthat emits light in the range of 420-460 nm which has, e.g., at least70% amino acid identity with the EBFP of SEQ ID NO: 150, at least 75%amino acid identity with the EBFP of SEQ ID NO: 150, at least 80% aminoacid identity with the EBFP of SEQ ID NO: 150, at least 85% amino acididentity with the EBFP of SEQ ID NO: 150, at least 90% amino acididentity with the EBFP of SEQ ID NO: 150 or at least 95% amino acididentity with the EBFP of SEQ ID NO: 150. In other aspects of thisembodiment, the fluorescent protein is a BFP that emits light in therange of 420-460 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the EBFP of SEQ ID NO: 150.

In yet another embodiment, the fluorescent protein is a yellowfluorescent protein. As used herein, the term “yellow fluorescentprotein” is synonymous with “YFP” and means a protein which absorbslight of a certain wavelength and emit peak light energy of wavelengthsin the range of 520-550 nm. Yellow fluorescent proteins useful in theinvention include, without limitation, the EYFP of SEQ ID NO: 151,genetically engineered EYFP variants and active EYFP fragments thereofthat retain the ability to emit peak light energy in the range of520-550 nm, the ZsYellow of SEQ ID NO: 152, genetically engineeredZsYellow variants and active ZsYellow fragments thereof that retain theability to emit peak light energy in the range of 520-550 nm, as wellas, naturally-occurring YFPs, naturally occurring YFP variants,genetically engineered YFP variants and active YFP fragments thereofthat retain the ability to emit peak light energy in the range of520-550 nm. As non-limiting examples, the YFP variants “Citrine,” whichcontain Val68Leu and Gln69Met substitutions in the YFP of SEQ ID NO:151, and “Venus,” which contain Phe46Leu, Met153Thr, Val163Ala andSer175Gly substitutions in the YFP of SEQ ID NO: 151, are extremelybright and fast-maturing YFPs, see, e.g., Oliver Griesbeck et al.,Reducing the environmental sensitivity of yellow fluorescent protein.Mechanism and applications, 276(31) J. Biol. Chem. 29188-29194 (2001);and Takeharu Nagai et al., A variant of yellow fluorescent protein withfast and efficient maturation for cell-biological applications, 20(1)Nat. Biotechnol. 87-90 (2002); and Aequorea-derived fluorescent proteinsas described in, e.g., Roger Y. Tsien et al., Long Wavelength EngineeredFluorescent Proteins, U.S. Pat. No. 6,124,128 (Sep. 26, 2000), U.S. Pat.No. 6,054,321 (Apr. 25, 2000), U.S. Pat. No. 6,077,707 (Jun. 20, 2000),U.S. Pat. No. 6,403,374 (Jun. 11, 2002) and U.S. Pat. No. 6,780,975(Aug. 24, 2004).

Thus, in aspects of this embodiment, the fluorescent protein is a YFPthat emits light in the range of 520-550 nm which has, e.g., at least70% amino acid identity with the YFP of SEQ ID NO: 151, at least 75%amino acid identity with the YFP of SEQ ID NO: 151, at least 80% aminoacid identity with the YFP of SEQ ID NO: 151, at least 85% amino acididentity with the YFP of SEQ ID NO: 151, at least 90% amino acididentity with the YFP of SEQ ID NO: 151 or at least 95% amino acididentity with the YFP of SEQ ID NO: 151. In other aspects of thisembodiment, the fluorescent protein is a YFP that emits light in therange of 520-550 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the YFP of SEQ ID NO: 151.

In other aspects of this embodiment, the fluorescent protein is a YFPthat emits light in the range of 520-550 nm which has, e.g., at least70% amino acid identity with the ZsYellow of SEQ ID NO: 152, at least75% amino acid identity with the ZsYellow of SEQ ID NO: 152, at least80% amino acid identity with the ZsYellow of SEQ ID NO: 152, at least85% amino acid identity with the ZsYellow of SEQ ID NO: 152, at least90% amino acid identity with the ZsYellow of SEQ ID NO: 152 or at least95% amino acid identity with the ZsYellow of SEQ ID NO: 152. In stillother aspects of this embodiment, the fluorescent protein is a YFP thatemits light in the range of 520-550 nm which has, e.g., at most one,two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the ZsYellow of SEQ ID NO: 152.

In yet embodiment, the fluorescent protein is a red fluorescent protein.As used herein, the term “red fluorescent protein” is synonymous with“RFP” and means a protein which absorbs light of a certain wavelengthand emit peak light energy of wavelengths in the range of 550-740 nm.Red fluorescent proteins useful in the invention include, withoutlimitation, the Discosoma striate RFP DsRed of SEQ ID NO: 153, DsRed1 ofSEQ ID NO: 154, DsRed2 of SEQ ID NO: 155 and DsRed Express of SEQ ID NO:156, genetically engineered DsRed, DsRed1, DsRed2 and DsRed Expressvariants and active DsRed, DsRed1, DsRed2 and DsRed Express fragmentsthereof that retain the ability to emit peak light energy in the rangeof 550-740 nm; the Heteractis crispa RFP HcRed of SEQ ID NO: 157,genetically engineered HcRed variants and active HcRed fragments thereofthat retain the ability to emit peak light energy in the range of550-740 nm; the Anemonia sulcata RFP AsRed of SEQ ID NO: 158,genetically engineered AsRed variants and active AsRed fragments thereofthat retain the ability to emit peak light energy in the range of550-740 nm, as well as, naturally-occurring RFPs, naturally occurringRFP variants, genetically engineered RFP variants and active RFPfragments thereof that retain the ability to emit peak light energy inthe range of 550-740 nm. As a non-limiting example, Entacmeaequadricolor fluorescent proteins including red fluorescent proteins suchas, e.g., eqFP611, see, e.g., Jörg Wiedenmann et al., A far-redfluorescent protein with fast maturation and reduced oligomerizationtendency from Entacmaea quadricolor (Anthozoa, Actinaria), 99(18) Proc.Natl. Acad. Sci. U.S.A. 11646-11651 (2002).

Thus, in aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the DsRed of SEQ ID NO: 153, at least75% amino acid identity with the DsRed of SEQ ID NO: 153, at least 80%amino acid identity with the DsRed of SEQ ID NO: 153, at least 85% aminoacid identity with the DsRed of SEQ ID NO: 153, at least 90% amino acididentity with the DsRed of SEQ ID NO: 153 or at least 95% amino acididentity with the DsRed of SEQ ID NO: 153. In other aspects of thisembodiment, the fluorescent protein is a RFP that emits light in therange of 550-740 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the DsRed of SEQ ID NO: 153.

In other aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the DsRed1 of SEQ ID NO: 154, atleast 75% amino acid identity with the DsRed1 of SEQ ID NO: 154, atleast 80% amino acid identity with the DsRed1 of SEQ ID NO: 154, atleast 85% amino acid identity with the DsRed1 of SEQ ID NO: 154, atleast 90% amino acid identity with the DsRed1 of SEQ ID NO: 154 or atleast 95% amino acid identity with the DsRed1 of SEQ ID NO: 154. Instill other aspects of this embodiment, the fluorescent protein is a RFPthat emits light in the range of 550-740 nm which has, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the DsRed1 of SEQ ID NO: 154.

In other aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the DsRed2 of SEQ ID NO: 155, atleast 75% amino acid identity with the DsRed2 of SEQ ID NO: 155, atleast 80% amino acid identity with the DsRed2 of SEQ ID NO: 155, atleast 85% amino acid identity with the DsRed2 of SEQ ID NO: 155, atleast 90% amino acid identity with the DsRed2 of SEQ ID NO: 155 or atleast 95% amino acid identity with the DsRed2 of SEQ ID NO: 155. Instill other aspects of this embodiment, the fluorescent protein is a RFPthat emits light in the range of 550-740 nm which has, e.g., at mostone, two, three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the DsRed2 of SEQ ID NO: 155.

In other aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the DsRed2 of SEQ ID NO: 156, atleast 75% amino acid identity with the DsRed Express of SEQ ID NO: 156,at least 80% amino acid identity with the DsRed Express of SEQ ID NO:156, at least 85% amino acid identity with the DsRed Express of SEQ IDNO: 156, at least 90% amino acid identity with the DsRed Express of SEQID NO: 156 or at least 95% amino acid identity with the DsRed Express ofSEQ ID NO: 156. In still other aspects of this embodiment, thefluorescent protein is a RFP that emits light in the range of 550-740 nmwhich has, e.g., at most one, two, three, four, five, six, seven, eight,nine, or ten amino acid substitutions relative to the DsRed Express ofSEQ ID NO: 156.

In other aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the AsRed of SEQ ID NO: 158, at least75% amino acid identity with the AsRed of SEQ ID NO: 158, at least 80%amino acid identity with the AsRed of SEQ ID NO: 158, at least 85% aminoacid identity with the AsRed of SEQ ID NO: 158, at least 90% amino acididentity with the AsRed of SEQ ID NO: 158 or at least 95% amino acididentity with the AsRed of SEQ ID NO: 158. In still other aspects ofthis embodiment, the fluorescent protein is a RFP that emits light inthe range of 550-740 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the AsRed of SEQ ID NO: 158.

In other aspects of this embodiment, the fluorescent protein can be aRFP that emits light in the range of 550-740 nm which has, e.g., atleast 70% amino acid identity with the HcRed of SEQ ID NO: 157, at least75% amino acid identity with the HcRed of SEQ ID NO: 157, at least 80%amino acid identity with the HcRed of SEQ ID NO: 157, at least 85% aminoacid identity with the HcRed of SEQ ID NO: 157, at least 90% amino acididentity with the HcRed of SEQ ID NO: 157 or at least 95% amino acididentity with the HcRed of SEQ ID NO: 157. In still other aspects ofthis embodiment, the fluorescent protein is a RFP that emits light inthe range of 550-740 nm which has, e.g., at most one, two, three, four,five, six, seven, eight, nine, or ten amino acid substitutions relativeto the HcRed of SEQ ID NO: 157.

TABLE 9 Excitation and Emission Maxima of Exemplary Fluorescent ProteinsFluorescent protein Excitation maxima (nm) Emission maxima (nm) EBFP 380440 ECFP 439 476 AmCyan 458 489 AcGFP 475 505 ZsGreen 493 505 Vitality ®hrGFP 500 506 EGFP 484 510 Monster Green ® 505 515 EYFP 512 529 ZsYellow529 539 DsRed-Express 557 579 DsRed2 563 582 DsRed 558 583 AsRed2 576592 HcRed1 588 618

Clostridial toxin substrates disclosed in the present specificationinclude, in part, a fluorophore binding protein. As used herein, theterm “fluorophore binding protein” means a peptide that can covalentlyor non-covalently associate with a fluorophore. A fluorophore bindingprotein can establish a covalent bond, or strong non-covalentinteraction, with a fluorophore in a selective chemical or biochemicalreaction. Nonlimiting examples of such fluorophore binding proteins andcorresponding fluorophores include the bis-arsenical tetracysteinesystem, see, e.g., B. Albert Griffin et al., Specific covalent labelingof recombinant protein molecules inside live cells, 281(5374) Science269-272 (1998); and B. Albert Griffin et al., Fluorescent labeling ofrecombinant proteins in living cells with FlAsH, 327 Methods Enzymol.565-578 (2000); the alkylguanine-DNA-alkyltransferase (AGT) system, see,e.g., Antje Keppler et al, A General Method for the Covalant Labeling ofFusion proteins with Small Molecules in vivo, 21(1) Nat. Biotech 86-89(2003); Antje Keppler et al, Labeling of fusion proteins ofO6-alkylguanine-DNA alkyltransferase with small molecules in vivo and invitro, 32(4) Methods 437-444 (2004); and Antje Keppler et al, Labelingof Fusion Proteins with Synthetic Fluorophores in Live Cells, 101(27)Proc. Natl. Acad. Sci. USA 9955-9959 (2004); and the dehalogenasesystem. In addition, non-limiting examples of fluorophore bindingproteins and corresponding fluorophores, as well as well-characterizedreagents, conditions and protocols are readily available from commercialvendors that include, without limitation, TC-FlAsH™ TC-ReAsH™ In-CellTetracysteine Tag Detection Kit (Invitrogin Corp., Carlsbad, Calif.);SNAP-tag™ multi-purpose protein tag system (Covalys Biosciences AG,Switzerland); and HaloTag™ Interchangeable Labeling Technology (PromegaCorp., Madison Wis.). These protocols are routine procedures well withinthe scope of one skilled in the art and from the teaching herein.

TABLE 10 Excitation and Emission Maxima of Exemplary Fluorophores forFluorophore Binding Proteins Name Dye Excitation maxima (nm) Emissionmaxima (nm) bis-Arsenical Tetracysteine System FlAsH fluoresceinarsenical hairpin binding dye 508 528 ReAsH resorufin arsenical hairpinbinding dye 593 608 AGT/SNAP-Tag System BG-430 para-benzyl guaninediethylaminocoumarin 421 444 and 484 BG-DAF para-benzyl guaninediacetylfluorescein 500 524 BG-505 para-benzyl guanine dyomic DY-505-05504 532 BG-488 para-benzyl guanine ATTO 488 506 526 BG-532 para-benzylguanine ATTO 532 536 554 BG-547 para-benzyl guanine dyomic DY-547 554568 TMR-Star para-benzyl guanine tetramethylrhodamine 554 580 BG-600para-benzyl guanine ATTO 600 606 626 BG-632 para-benzyl guanine dyomicDY-632 636 656 BG-647 para-benzyl guanine dyomic DY-647 660 673 BG-732para-benzyl guanine dyomic DY-732 732 747 BG-747 para-benzyl guaninedyomic DY-747 752 763 Dehalogenase/HaloTag ™ System HaloTag Coumarianderivative 353 434 Coumarian HaloTag nonfluorescent diacetyl fluorescein494 526 diAcFAM derivative HaloTag TMR tetramethyl rhodamine derivative555 585

The bis-arsenical tetracysteine system comprises a fusion proteinincluding the protein of interest and a tetracysteine hexapeptidecomprising the amino acid sequence C-C-X-X-C-C (SEQ ID NO: 182) and abis-arsenical fluorophore complexed with two dithiol residues. In thelabeling reaction, the tetracysteine peptide displaces the dithiols fromthe arsenic residues of the fluorophore. This interaction stronglycouples the fluorophore with the fluorophore binding protein andsignificantly increases the signal by reducing the quenching of thefluorophore. Nonlimiting examples of bis-arsenical fluorophores includenonfluorescent biarsenical derivitives of fluorescein, such as, e.g.,FlAsH and nonfluorescent biarsenical derivitives of resorufin, such as,e.g., ReAsH.

The AGT system comprises a fusion protein including the protein ofinterest and a modified AGT 22 kDa polypeptide (SEQ ID NO: 183) and abenzyl guanine modified in the para-position by a fluorescent label. Inthe labeling reaction, the 06-position of the para-substituted benzylguanine irreversibly binds to a reactive csyteine in the active centerof AGT. Nonlimiting examples of modified benzylguanine fluorophoreslisted in Table 10.

The dehalogenase system comprises a fusion protein including the proteinof interest and a modified dehalogenase and a modified fluorophorecomprising an alkyl residue. In the labeling reaction, the modifiedfluorophore strongly interacts with the active site of the modifieddehalogenase. The modified dehalogenase is a 33 kDa polypeptide (SEQ IDNO: 184) comprising a mutation in the active center that significantlyslows the catalytic activity of the enzyme, effectively creating anirreversible interaction. Nonlimiting examples of modified benzylguaninefluorophores listed in Table 10.

Thus, in an embodiment, a fluorophore binding protein is a tetracysteinepeptide which strongly interacts with a fluorophore. In an aspect ofthis embodiment, the fluorophore binding protein is a tetracysteinepeptide comprises SEQ ID NO: 182 which strongly interacts with afluorophore. In another aspect of this embodiment, the fluorophorebinding protein is a tetracysteine peptide that strongly interacts witha nonfluorescent biarsenical derivitives of fluorescein. In anotheraspect of this embodiment, the fluorophore binding protein is atetracysteine peptide that strongly interacts with a nonfluorescentbiarsenical derivitives of resorufin.

Thus, in an embodiment, a fluorophore binding protein is an AGTpolypeptide which strongly interacts with a fluorophore. In an aspect ofthis embodiment, the fluorophore binding protein is an AGT whichstrongly interacts with a fluorophore comprises SEQ ID NO: 183. In otheraspects of this embodiment, the fluorophore binding protein can be a AGTwhich strongly interacts with a fluorophore that has, e.g., at least 70%amino acid identity with the AGT of SEQ ID NO: 183, at least 75% aminoacid identity with the AGT of SEQ ID NO: 183, at least 80% amino acididentity with the AGT of SEQ ID NO: 183, at least 85% amino acididentity with the AGT of SEQ ID NO: 183, at least 90% amino acididentity with the AGT of SEQ ID NO: 183 or at least 95% amino acididentity with the AGT of SEQ ID NO: 183. In still other aspects of thisembodiment, the fluorophore binding protein is a AGT which stronglyinteracts with a fluorophore that has, e.g., at most one, two, three,four, five, six, seven, eight, nine, or ten amino acid substitutionsrelative to the AGT of SEQ ID NO: 183. In other aspects of thisembodiment, the fluorophore binding protein is an AGT that stronglyinteracts with a para-substituted benzyl guanine derivitive comprising adiethylaminocoumarin, a diacetylfluorescein, a dyomic DY-505-05, an ATTO488, an ATTO 532, a DY-547, a tetramethylrhodamine, an ATTO 600, adyomic DY-632, a dyomic DY-647, a dyomic DY-732 or a dyomic DY-747.

Thus, in an embodiment, a fluorophore binding protein is a dehalogenasepolypeptide which strongly interacts with a fluorophore. In an aspect ofthis embodiment, the fluorophore binding protein is a dehalogenase whichstrongly interacts with a fluorophore comprises SEQ ID NO: 184. In otheraspects of this embodiment, the fluorophore binding protein can be adehalogenase which strongly interacts with a fluorophore that has, e.g.,at least 70% amino acid identity with the dehalogenase of SEQ ID NO:184, at least 75% amino acid identity with the dehalogenase of SEQ IDNO: 184, at least 80% amino acid identity with the dehalogenase of SEQID NO: 184, at least 85% amino acid identity with the dehalogenase ofSEQ ID NO: 184, at least 90% amino acid identity with the dehalogenaseof SEQ ID NO: 184 or at least 95% amino acid identity with thedehalogenase of SEQ ID NO: 184. In still other aspects of thisembodiment, the fluorophore binding protein is a dehalogenase whichstrongly interacts with a fluorophore that has, e.g., at most one, two,three, four, five, six, seven, eight, nine, or ten amino acidsubstitutions relative to the dehalogenase of SEQ ID NO: 184. In otheraspects of this embodiment, the fluorophore binding protein is andehalogenase that strongly interacts with a coumarian derivitive such asHaloTag Coumarian, a fluorescein derivitive such as HaloTag diAcFAM or atetramethyl rhodamine derivitive such as HaloTag TMR.

It is understood that a Clostridial toxin substrate useful in theinvention optionally can include one or more additional components. As anon-limiting example, a flexible spacer sequence such as GGGGS (SEQ IDNO: 159) can be included in a Clostridial toxin substrate useful in theinvention. A useful Clostridial toxin substrate further can include,without limitation, one or more of the following: epitope-binding tags,such as. e.g., FLAG, Express™, human Influenza virus hemagluttinin (HA),human p62^(c-Myc) protein (c-MYC), Vesicular Stomatitis VirusGlycoprotein (VSV-G), glycoprotein-D precursor of Herpes simplex virus(HSV), V5, and AU1; affinity-binding, such as. e.g., polyhistidine(HIS), streptavidin binding peptide (strep), and biotin or abiotinylation sequence; peptide-binding regions, such as. e.g., theglutathione binding domain of glutathione-S-transferase, the calmodulinbinding domain of the calmodulin binding protein, and the maltosebinding domain of the maltose binding protein; immunoglobulin hingeregion; an N-hydroxysuccinimide linker; a peptide or peptidomimetichairpin turn; or a hydrophilic sequence or another component or sequencethat, for example, promotes the solubility or stability of theClostridial toxin substrate. Non-limiting examples of specific protocolsfor selecting, making and using an appropriate binding peptide aredescribed in, e.g., Epitope Tagging, pp. 17.90-17.93 (Sambrook andRussell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed.2001); Antibodies: A Laboratory Manual (Edward Harlow & David Lane,eds., Cold Spring Harbor Laboratory Press, 2^(nd) ed. 1998); and UsingAntibodies: A Laboratory Manual: Portable Protocol No. I (Edward Harlow& David Lane, Cold Spring Harbor Laboratory Press, 1998), which arehereby incorporated by reference. In addition, non-limiting examples ofbinding peptides as well as well-characterized reagents, conditions andprotocols are readily available from commercial vendors that include,without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BDBiosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Aspects of the present invention provide compositions comprising a cellthat contains an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of said cell wherein said cell iscapable of Clostridial toxin intoxication, and wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain.

Other aspects of the present invention provide compositions comprising acell that transiently contains an exogenous Clostridial toxin substratecapable of being localized to the plasma membrane of said cell whereinsaid cell is capable of Clostridial toxin intoxication, wherein saidsubstrate comprises a fluorescent member, a membrane targeting domainand a Clostridial toxin recognition sequence comprising a cleavage site,where the cleavage site intervenes between said fluorescent member andsaid membrane localization domain.

Other aspects of the present invention provide compositions comprising acell that stably contains an exogenous Clostridial toxin substratecapable of being localized to the plasma membrane of said cell whereinsaid cell is capable of Clostridial toxin intoxication, and wherein saidsubstrate comprises a fluorescent member, a membrane targeting domainand a Clostridial toxin recognition sequence comprising a cleavage site,where the cleavage site intervenes between said fluorescent member andsaid membrane localization domain.

Yet other aspects of the present invention provide compositionscomprising a cell population, the cell population comprising cells thatcontain an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of said cells wherein said cells arecapable of Clostridial toxin intoxication, and wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain and wherein greater than 50% of said cellpopulation comprises said cells containing said exogenous Clostridialtoxin substrate.

Yet other aspects of the present invention provide compositionscomprising a cell population, the cell population comprising cells thattransiently contain an exogenous Clostridial toxin substrate capable ofbeing localized to the plasma membrane of said cells wherein said cellsare capable of Clostridial toxin intoxication, wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain and wherein greater than 50% of said cellpopulation comprises said cells containing said exogenous Clostridialtoxin substrate.

Yet other aspects of the present invention provide compositionscomprising a cell population, said cell population comprising cells thatstably contain an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of said cells wherein said cells arecapable of Clostridial toxin intoxication, and wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain.

The cell compositions disclosed in the present specification include, inpart, a cell capable of Clostridial toxin intoxication. As used herein,the term “cell,” means any eukaryotic cell that expresses, or can beengineered to express, at least one receptor that binds a Clostridialtoxin. The term cell encompasses cells from a variety of organisms, suchas, e.g., murine, rat, porcine, bovine, equine, primate and human cells;from a variety of cell types such as, e.g., neural and non-neural; andcan be isolated from or part of a heterogeneous cell population, tissueor organism. It is understood that cells useful in aspects of theinvention can included, without limitation, primary cells; culturedcells; established cells; normal cells; transformed cells; tumor cells;infected cells; proliferating and terminally differentiated cells; andstably or transiently transfected cells, including stably andtransiently transfected cells. It is further understood that cellsuseful in aspects of the invention can be in any state such asproliferating or quiescent; intact or permeabilized such as throughchemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-laminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated and polybrene-mediated;physical-mediated transfection, such as, e.g., biolistic particledelivery, microinjection and electroporation; and viral-mediatedtransfection, such as, e.g., retroviral-mediated transfection. It isfurther understood that cells useful in aspects of the invention mayinclude those which express a Clostridial toxin substrate under controlof a constitutive, tissue-specific, cell-specific or inducible promoterelement, enhancer element or both. It further is understood that cellsuseful in aspects of the invention may or may not express one or moreendogenous Clostridial toxin target proteins such as, e.g., SNAP-25,VAMP and syntaxin.

As used herein, the term “cell capable of Clostridial toxinintoxication” means a cell that can enable the overall cellularmechanism whereby a Clostridial toxin proteolytically cleaves asubstrate and encompasses the binding of a Clostridial toxin to a low orhigh affinity receptor, the internalization of the toxin/receptorcomplex, the translocation of the Clostridial toxin light chain into thecytoplasm and the enzymatic target modification of a Clostridial toxinsubstrate. By definition, a cell capable of Clostridial toxinintoxication must express one or more endogenous low or high affinityClostridial toxin receptors for one or more Clostridial toxin serotypes;express one or more exogenous low or high affinity Clostridial toxinreceptors for one or more Clostridial toxin serotypes; or express acombination of endogenous low or high affinity Clostridial toxinreceptors and exogenous low or high affinity Clostridial toxin receptorsfor one or more Clostridial toxin serotypes.

Thus, in an embodiment, a cell capable of Clostridial toxin intoxicationcan be a cell expressing a Clostridial toxin receptor. In aspects ofthis embodiment, the Clostridial toxin receptor can be a low affinityClostridial toxin receptor, a high affinity Clostridial toxin receptor,an endogenous Clostridial toxin receptor, an exogenous Clostridial toxinreceptor, or any combination thereof. In other aspects of thisembodiment, the Clostridial toxin receptor can be a BoNT/A receptor, aBoNT/B receptor, a BoNT/C1 receptor, a BoNT/D receptor, a BoNT/Ereceptor, a BoNT/F receptor, a BoNT/G receptor or a TeNT receptor.

In another embodiment, a cell capable of Clostridial toxin intoxicationcan be a cell expressing a plurality of Clostridial toxin receptors. Inaspects of this embodiment, a plurality of Clostridial toxin receptorcan comprise low affinity Clostridial toxin receptors, high affinityClostridial toxin receptors, endogenous Clostridial toxin receptors,exogenous Clostridial toxin receptors, or any combination thereof. Inaspects of this embodiment, a plurality of Clostridial toxin receptorcan comprise, e.g., two or more Clostridial toxin receptors, three ormore Clostridial toxin receptors, four or more Clostridial toxinreceptors, five or more Clostridial toxin receptors, six or moreClostridial toxin receptors, seven or more Clostridial toxin receptorsand eight or more Clostridial toxin receptors. In other aspects of thisembodiment, cell capable of Clostridial toxin intoxication can expresstwo or more of the following receptors a BoNT/A receptor, a BoNT/Breceptor, a BoNT/C1 receptor, a BoNT/D receptor, a BoNT/E receptor, aBoNT/F receptor, a BoNT/G receptor or a TeNT receptor. In other aspectsof this embodiment, cell capable of Clostridial toxin intoxication canexpress three or more of the following receptors a BoNT/A receptor, aBoNT/B receptor, a BoNT/C1 receptor, a BoNT/D receptor, a BoNT/Ereceptor, a BoNT/F receptor, a BoNT/G receptor or a TeNT receptor. Inother aspects of this embodiment, cell capable of Clostridial toxinintoxication can express four or more of the following receptors aBoNT/A receptor, a BoNT/B receptor, a BoNT/C1 receptor, a BoNT/Dreceptor, a BoNT/E receptor, a BoNT/F receptor, a BoNT/G receptor or aTeNT receptor. In other aspects of this embodiment, cell capable ofClostridial toxin intoxication can express five or more of the followingreceptors a BoNT/A receptor, a BoNT/B receptor, a BoNT/C1 receptor, aBoNT/D receptor, a BoNT/E receptor, a BoNT/F receptor, a BoNT/G receptoror a TeNT receptor. In other aspects of this embodiment, cell capable ofClostridial toxin intoxication can express six or more of the followingreceptors a BoNT/A receptor, a BoNT/B receptor, a BoNT/C1 receptor, aBoNT/D receptor, a BoNT/E receptor, a BoNT/F receptor, a BoNT/G receptoror a TeNT receptor. In other aspects of this embodiment, cell capable ofClostridial toxin intoxication can express seven or more of thefollowing receptors a BoNT/A receptor, a BoNT/B receptor, a BoNT/C1receptor, a BoNT/D receptor, a BoNT/E receptor, a BoNT/F receptor, aBoNT/G receptor or a TeNT receptor.

Cells that express one or more endogenous or exogenous Clostridial toxinreceptors can be identified by routine methods including direct andindirect assays for toxin uptake. Assays that determine Clostridialtoxin binding or uptake properties can be used to assess whether a cellis expressing a Clostridial toxin receptor. Such assays include, withoutlimitation, cross-linking assays using labeled Clostridial toxins, suchas, e.g., [¹²⁵I] BoNT/A, [¹²⁵I] BoNT/B, [¹²⁵I] BoNT/C1, [¹²⁵I] BoNT/D,[¹²⁵I] BoNT/E, [¹²⁵I] BoNT/F, [¹²⁵I] BoNT/G and [¹²⁵I] TeNT, see, e.g.,Noriko Yokosawa et al., Binding of Clostridium botulinum type Cneurotoxin to different neuroblastoma cell lines, 57(1) Infect. Immun.272-277 (1989); Noriko Yokosawa et al., Binding of botulinum type Cl, Dand E neurotoxins to neuronal cell lines and synaptosomes, 29(2) Toxicon261-264 (1991); and Tei-ichi Nishiki et al., Identification of proteinreceptor for Clostridium botulinum type B neurotoxin in rat brainsynaptosomes, 269(14) J. Biol. Chem. 10498-10503 (1994). Othernon-limiting assays include immunocytochemical assays that detect toxinbinding using labeled or unlabeled antibodies, see, e.g., AtsushiNishikawa et al., The receptor and transporter for internalization ofClostridium botulinum type C progenitor toxin into HT-29 cells, 319(2)Biochem. Biophys. Res. Commun. 327-333 (2004) and immunoprecipitationassays, see, e.g., Yukako Fujinaga et al., Molecular characterization ofbinding subcomponents of Clostridium botulinum type C progenitor toxinfor intestinal epithelial cells and erythrocytes, 150 (Pt 5)Microbiology 1529-1538 (2004), that detect bound toxin using labeled orunlabeled antibodies. Antibodies useful for these assays include,without limitation, antibodies selected against a Clostridial toxin,such as, e.g., BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/Gor TeNT, antibodies selected against a CoNT receptor, such as, e.g.,FGFR3 or synaptotagmin, and/or antibodies selected against aganglioside, such as, e.g., GD1a, GD1b, GD3, GQ1b, or GT1b. If theantibody is labeled, the binding of the molecule can be detected byvarious means, including Western blotting, direct microscopicobservation of the cellular location of the antibody, measurement ofcell or substrate-bound antibody following a wash step, orelectrophoresis, employing techniques well-known to those of skill inthe art. If the antibody is unlabeled, one may employ a labeledsecondary antibody for indirect detection of the bound molecule, anddetection can proceed as for a labeled antibody. It is understood thatthese and similar assays that determine Clostridial toxin uptakeproperties or characteristics can be useful in selecting a neuron orother cells useful in aspects of the invention.

Assays that monitor the release of a molecule after exposure to aClostridial toxin can also be used to assess whether a cell isexpressing a Clostridial toxin receptor. In these assays, inhibition ofthe molecule's release would occur in cells expressing a Clostridialtoxin receptor after Clostridial toxin treatment. Well known assaysinclude methods that measure inhibition of radio-labeled catecholaminerelease from neurons, such as, e.g., [³H] noradrenaline or [³H] dopaminerelease, see e.g., A Fassio et al., Evidence for calcium-dependentvesicular transmitter release insensitive to tetanus toxin and botulinumtoxin type F, 90(3) Neuroscience 893-902 (1999); and Sara Stigliani etal., The sensitivity of catecholamine release to botulinum toxin C1 andE suggests selective targeting of vesicles set into the readilyreleasable pool, 85(2) J. Neurochem. 409-421 (2003), or measurescatecholamine release using a fluorometric procedure, see, e.g., Antonde Paiva et al., A role for the interchain disulfide or itsparticipating thiols in the internalization of botulinum neurotoxin Arevealed by a toxin derivative that binds to ecto-acceptors and inhibitstransmitter release intracellularly, 268(28) J. Biol. Chem. 20838-20844(1993); Gary W. Lawrence et al., Distinct exocytotic responses of intactand permeabilised chromaffin cells after cleavage of the 25-kDasynaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinumtoxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and Patrick Foranet al., Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 inintact and permeabilized chromaffin cells: correlation with its blockadeof catecholamine release, 35(8) Biochemistry 2630-2636 (1996). Othernon-limiting examples include assays that measure inhibition of hormonerelease from endocrine cells, such as, e.g., anterior pituitary cells orovarian cells. It is understood that these and similar assays formolecule release can be useful in selecting a neuron or other cellsuseful in aspects of the invention.

Assays that detect the cleavage of a Clostridial toxin substrate afterexposure to a Clostridial toxin can also be used to assess whether acell is expressing a Clostridial toxin receptor. In these assays,generation of a Clostridial toxin substrate cleavage-product would bedetected in cells expressing a Clostridial toxin receptor afterClostridial toxin treatment. Non-limiting examples of specific Westernblotting procedures, as well as well-characterized reagents, conditionsand protocols are readily available from commercial vendors thatinclude, without limitation, Amersham Biosciences, Piscataway, N.J.;Bio-Rad Laboratories, Hercules, Calif.; Pierce Biotechnology, Inc.,Rockford, Ill.; Promega Corporation, Madison, Wis., and Stratagene,Inc., La Jolla, Calif. It is understood that these and similar assaysfor Clostridial toxin substrate cleavage can be useful in selecting aneuron or other cells useful in aspects of the invention.

As non-limiting examples, western blot analysis using an antibody thatspecifically recognizes BoNT/A SNAP-25-cleaved product can be used toassay for uptake of BoNT/A; western blot analysis using an antibody thatspecifically recognizes BoNT/C1 SNAP-25-cleaved product can be used toassay for uptake of BoNT/C1; and western blot analysis using an antibodythat specifically recognizes a BoNT/E SNAP-25-cleaved product can beused to assay for uptake of BoNT/E. Examples of anti-SNAP-25 antibodiesuseful for these assays include, without limitation, rabbit polyclonalanti-SNAP25₁₉₇ antiserum pAb anti-SNAP25₁₉₇ #1 (Allergan, Inc., Irvine,Calif.), mouse monoclonal anti-SNAP-25 antibody SMI-81 (SternbergerMonoclonals, Lutherville, Md.), mouse monoclonal anti-SNAP-25 antibodyCI 71.1 (Synaptic Systems, Goettingen, Germany), mouse monoclonalanti-SNAP-25 antibody CI 71.2 (Synaptic Systems, Goettingen, Germany),mouse monoclonal anti-SNAP-25 antibody SP12 (Abcam, Cambridge, Mass.),rabbit polyclonal anti-SNAP-25 antiserum (Synaptic Systems, Goettingen,Germany), and rabbit polyclonal anti-SNAP-25 antiserum (Abcam,Cambridge, Mass.).

As additional non-limiting examples, western blot analysis using anantibody that specifically recognizes a BoNT/B VAMP-cleaved product canbe used to assay for uptake of BoNT/B; western blot analysis using anantibody that specifically recognizes BoNT/D VAMP-cleaved product can beused to assay for uptake of BoNT/D; western blot analysis using anantibody that specifically recognizes BoNT/F VAMP-cleaved product can beused to assay for uptake of BoNT/F; western blot analysis using anantibody that specifically recognizes BoNT/G VAMP-cleaved product can beused to assay for uptake of BoNT/G; and western blot analysis using anantibody that specifically recognizes TeNT. Examples of anti-VAMPantibodies useful for these assays include, without limitation, mousemonoclonal anti-VAMP-1 antibody CI 10.1 (Synaptic Systems, Goettingen,Germany), mouse monoclonal anti-VAMP-1 antibody SP10 (Abcam, Cambridge,Mass.), mouse monoclonal anti-VAMP-1 antibody SP11 (Abcam, Cambridge,Mass.), rabbit polyclonal anti-VAMP-1 antiserum (Synaptic Systems,Goettingen, Germany), rabbit polyclonal anti-VAMP-1 antiserum (Abcam,Cambridge, Mass.), mouse monoclonal anti-VAMP-2 antibody CI 69.1(Synaptic Systems, Goettingen, Germany), rabbit polyclonal anti-VAMP-2antiserum (Synaptic Systems, Goettingen, Germany), rabbit polyclonalanti-VAMP-2 antiserum (Abcam, Cambridge, Mass.), mouse monoclonalanti-VAMP-3 antibody CI 10.1 (Synaptic Systems, Goettingen, Germany),rabbit polyclonal anti-VAMP-3 antiserum (Synaptic Systems, Goettingen,Germany) and rabbit polyclonal anti-VAMP-3 antiserum (Abcam, Cambridge,Mass.),

As another non-limiting example, western blot analysis using an antibodythat specifically recognizes BoNT/C1 Syntaxin-cleaved product can beused to assay for uptake of BoNT/C1. Examples of anti-Syntaxinantibodies useful for these assays include, without limitation, mousemonoclonal anti-Syntaxin-1 antibody CI 78.2 (Synaptic Systems,Goettingen, Germany), mouse monoclonal anti-Syntaxin-1A antibody CI 78.3(Synaptic Systems, Goettingen, Germany), rabbit polyclonalanti-Syntaxin-1A antiserum (Synaptic Systems, Goettingen, Germany),rabbit polyclonal anti-Syntaxin-1B antiserum (Synaptic Systems,Goettingen, Germany), rabbit polyclonal anti-Syntaxin antiserum (Abcam,Cambridge, Mass.), rabbit polyclonal anti-Syntaxin-2 antiserum (Abcam,Cambridge, Mass.) and rabbit polyclonal anti-Syntaxin-3 antiserum(Abcam, Cambridge, Mass.),

It is envisioned that an exogenous Clostridial toxin receptor caninclude, without limitation, a nucleic acid molecule, such as, e.g., DNAand RNA, that encodes a Clostridial toxin receptor disclosed in thepresent specification and peptide molecule or peptidomimetic comprisinga Clostridial toxin receptor disclosed in the present specification. Inis also envisioned that an exogenous Clostridial toxin receptor can betransiently or stably expressed in a cell useful in aspects of theinvention. Thus, aspects of this embodiment include a cell thattransiently contains a nucleic acid molecule, such as, e.g., DNA andRNA, that encode a Clostridial toxin receptor disclosed in the presentspecification and a cell that transiently contains a peptide molecule orpeptidomimetic comprising Clostridial toxin receptor disclosed in thepresent specification. Other aspects of this embodiment include a cellthat stably contains a nucleic acid molecule, such as, e.g., DNA andRNA, that encode a Clostridial toxin substrate disclosed in the presentspecification and a cell that stably contains a peptide molecule orpeptidomimetic comprising Clostridial toxin substrate disclosed in thepresent specification. Stably-maintained nucleic acid molecules may beextra-chromosomal and replicate autonomously, or they may be integratedinto the chromosomal material of the cell and replicatenon-autonomously.

It is understood that the selection of a cell depends, in part, on whichClostridial toxin is to be assayed. As a non-limiting example, to assayfor BoNT/A activity, one selects a cell that expresses or can beengineered to express a low or high affinity receptor for BoNT/A. As afurther example, to assay for BoNT/B activity, one selects a cell thatexpresses or can be engineered to express a low or high affinityreceptor for BoNT/B. As a still further example, to assay for BoNT/C1activity, one selects a cell that expresses or can be engineered toexpress a low or high affinity receptor for BoNT/C1. As a still furtherexample, to assay for BoNT/D activity, one selects a cell that expressesor can be engineered to express a low or high affinity receptor forBoNT/D. As a still further example, to assay for BoNT/E activity, oneselects a cell that expresses or can be engineered to express a low orhigh affinity receptor for BoNT/E. As a still further example, to assayfor BoNT/F activity, one selects a cell that expresses or can beengineered to express a low or high affinity receptor for BoNT/F. As astill further example, to assay for BoNT/G activity, one selects a cellthat expresses or can be engineered to express a low or high affinityreceptor for BoNT/G. As a still further example, to assay for TeNTactivity, one selects a cell that expresses or can be engineered toexpress a low or high affinity receptor for TeNT.

As discussed above, it is understood that a cell useful in the inventionexpresses endogenous or exogenous low or high affinity Clostridial toxinreceptors for one or more Clostridial toxins. Such a cell also generallyexhibits inhibition of exocytosis upon exposure to Clostridial toxinwith, for example, an IC₅₀ of less than 500 nM, less than 100 mM, lessthan 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, lessthan 0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM. In particular embodiments, the invention provides a neuroncontaining a BoNT/A substrate which exhibits inhibition of exocytosiswith an IC₅₀ of less than 500 nM, less than 100 mM, less than 50 nM,less than 5 nM, less than 0.5 nM, less than 0.05 nM, less than 0.005 nM,less than 0.0005 nM, less than 0.00005 nM or less than 0.000005 nM uponexposure to BoNT/A. In further embodiments, the invention provides aneuron containing a BoNT/B substrate which exhibits inhibition ofexocytosis with an IC₅₀ of less than 500 nM, less than 100 mM, less than50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, less than0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM upon exposure to BoNT/B. In other embodiments, the inventionprovides a neuron containing a BoNT/C1 substrate which exhibitsinhibition of exocytosis with an IC₅₀ of less than 500 nM, less than 100mM, less than 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05nM, less than 0.005 nM, less than 0.0005 nM, less than 0.00005 nM orless than 0.000005 nM upon exposure to BoNT/C1. In still furtherembodiments, the invention provides a neuron containing a BoNT/Dsubstrate which exhibits inhibition of exocytosis with an IC₅₀ of lessthan 500 nM, less than 100 mM, less than 50 nM, less than 5 nM, lessthan 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than 0.0005 nM,less than 0.00005 nM or less than 0.000005 nM upon exposure to BoNT/D.In additional embodiments, the invention provides a neuron containing aBoNT/E substrate which exhibits inhibition of exocytosis with an IC₅₀ ofless than 500 nM, less than 100 mM, less than 50 nM, less than 5 nM,less than 0.5 nM, less than 0.05 nM, less than 0.005 nM, less than0.0005 nM, less than 0.00005 nM or less than 0.000005 nM upon exposureto BoNT/E. In yet further embodiments, the invention provides a neuroncontaining a BoNT/F substrate which exhibits inhibition of exocytosiswith an IC₅₀ of less than 500 nM, less than 100 mM, less than 50 nM,less than 5 nM, less than 0.5 nM, less than 0.05 nM, less than 0.005 nM,less than 0.0005 nM, less than 0.00005 nM or less than 0.000005 nM uponexposure to BoNT/F. In further embodiments, the invention provides aneuron containing a BoNT/G substrate which exhibits inhibition ofexocytosis with an IC₅₀ of less than 500 nM, less than 100 mM, less than50 nM, less than 5 nM, less than 0.5 nM, less than 0.05 nM, less than0.005 nM, less than 0.0005 nM, less than 0.00005 nM or less than0.000005 nM upon exposure to BoNT/G. In still further embodiments, theinvention provides a neuron containing a TeNT substrate which exhibitsinhibition of exocytosis with an IC₅₀ of less than 500 nM, less than 100mM, less than 50 nM, less than 5 nM, less than 0.5 nM, less than 0.05nM, less than 0.005 nM, less than 0.0005 nM, less than 0.00005 nM orless than 0.000005 nM upon exposure to TeNT. It is understood that thesame neuron can express two or more receptors for different Clostridialtoxin serotypes, with the same or a different IC₅₀ for each individualtoxin serotype.

Cells useful in aspects of the invention include both neuronal andnon-neuronal cells. Neuronal cells useful in aspects of the inventioninclude, without limitation, primary neuronal cells; immortalized orestablished neuronal cells; transformed neuronal cells; neuronal tumorcells; stably and transiently transfected neuronal cells and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman neuronal cells. Non-limiting examples of neuronal cells useful inaspects of the invention include, e.g., peripheral neuronal cells, suchas, e.g., motor neurons and sensory neurons; and CNS neuronal cells,such as, e.g., spinal cord neurons like embryonic spinal cord neurons,dorsal root ganglia (DRG) neurons, cerebral cortex neurons, cerebellarneurons, hippocampal neurons and motor neurons. Neuronal cells useful inthe invention include, without limitation, those described herein belowor tabulated in Table 12. Such neuronal cells can be, for example,central nervous system (CNS) neurons; neuroblastoma cells; motorneurons, hippocampal neurons or cerebellar neurons and further can be,without limitation, Neuro-2A, SH-SY5Y, NG108-15, N1E-115 or SK-N-DZcells. The skilled person understands that these and additional primaryand established neurons can be useful in the cells and methods of theinvention.

Neurons useful in aspects of the invention include, without limitation,primary cultures such as primary cultures of embryonic dorsal rootganglion (DRG) neurons. As one example, primary cultures of embryonicrat DRG neurons are described in Mary J. Welch et al., Sensitivity ofembryonic rat dorsal root ganglia neurons to Clostridium botulinumneurotoxins, 38(2) Toxicon 245 258 (2000); and primary cultures of fetalspinal cord neurons, for example, primary cultures of murine fetalspinal cord neurons are described in Elaine A. Neale et al., Botulinumneurotoxin A blocks synaptic vesicle exocytosis but not endocytosis atthe nerve terminal, 147(6) J. Cell Biol. 1249-1260 (1999), and John A.Chaddock et al., Inhibition of vesicular secretion in both neuronal andnon-neuronal cells by a retargeted endopeptidase derivative ofClostridium botulinum neurotoxin type A, 68(5) Infect. Immun. 2587-2593(2000). Thus, in an embodiment, a cell capable of Clostridial toxinintoxication can be a neuron. In aspects of this embodiment, a neuroncan be a neuron from, e.g., a primary culture, an embryonic dorsal rootganglion primary culture or a fetal spinal cord primary culture. Asnon-limiting examples, cells useful for determining Clostridial toxinactivity according to a method disclosed in the present specificationcan include, a primary neuronal cell, such as, e.g., rat embryonicdorsal root ganglion (DRG) neurons or murine fetal spinal cord neurons,that include a Clostridial toxin substrate comprising a SNAP-25recognition sequence; such as, e.g., a BoNT/A recognition sequence or aBoNT/E recognition sequence; a primary neuronal cell, such as, e.g., ratembryonic dorsal root ganglion (DRG) neurons or murine fetal spinal cordneurons, that include a Clostridial toxin substrate comprising a VAMPrecognition sequence; such as, e.g., a BoNT/B recognition sequence or aTeNT recognition sequence; and a primary neuronal cell, such as, e.g.,rat embryonic dorsal root ganglion (DRG) neurons or murine fetal spinalcord neurons, that include a Clostridial toxin substrate comprising aSyntaxin recognition sequence; such as, e.g., a BoNT/C1 recognitionsequence.

Neuronal cell lines useful in aspects of the invention include, withoutlimitation, neuroblastoma cell lines, neuronal hybrid cell lines, spinalcord cell lines, central nervous system cell lines, cerebral cortex celllines, dorsal root ganglion cell lines, hippocampal cell lines andpheochromocytoma cell lines.

Neuroblastoma cell lines, such as, e.g., murine, rat, primate or humanneuroblastoma cell lines can be useful in aspects of the invention.Neuroblastoma cell lines useful in aspects of the invention include,without limitation, BE(2)-C (ATCC CRL-2268; ECACC 95011817), BE(2)-M17(ATCC CRL-2267; ECACC 95011816), C1300 (ECACC 93120817), CHP-212 (ATCCCRL-2273), CHP-126 (DSMZ ACC 304), IMR 32 (ATCC CRL-127; ECACC 86041809;DSMZ ACC 165), KELLY (ECACC 92110411; DSMZ ACC 355), LA-N-2, see, e.g.,Robert C. Seeger et al., Morphology, growth, chromosomal pattern andfibrinolytic activity of two new human neuroblastoma cell lines, 37(5)Cancer Res. 1364-1371 (1977); and G. J. West et al., Adrenergic,cholinergic, and inactive human neuroblastoma cell lines with theaction-potential Na+ ionophore, 37(5) Cancer Res. 1372-1376 (1977),MC-IXC (ATCC CRL-2270), MHH-NB-11 (DSMZ ACC 157), N18Tg2 (DSMZ ACC 103),N1E-115 (ATCC CCL-2263; ECACC 88112303), N4TG3 (DSMZ ACC 101), Neuro-2A(ATCC CCL-131; ECACC 89121404; DSMZ ACC 148), NB41A3 (ATCC CCL-147;ECACC 89121405), NS20Y (DSMZ ACC 94), SH-SY5Y (ATCC CRL-2266; ECACC94030304; DSMZ ACC 209), SIMA (DSMZ ACC 164), SK-N-DZ (ATCC CRL-2149;ECACC 94092305), SK-N-F1 (ATCC CRL-2142, ECACC 94092304), SK-N-MC (ATCCHTB-10, DSMZ ACC 203) and SK-N-SH (ATCC HTB-11, ECACC 86012802). Thus,in an embodiment, a cell capable of Clostridial toxin intoxication canbe a neuroblastoma cell. In aspects of this embodiment, a neuroblastomacell can be, e.g., BE(2)-C, BE(2)-M17, C1300, CHP-212, CHP-126, IMR 32,KELLY, LA-N-2, MC-IXC, MHH-NB-11, N18Tg2, N1E-115, N4TG3, Neuro-2A,NB41A3, NS20Y, SH-SY5Y, SIMA, SK-N-DZ, SK-N-F1, SK-N-MC and SK-N-SH. Asnon-limiting examples, cells useful for determining Clostridial toxinactivity according to a method disclosed in the present specificationcan include, a neuroblastoma cell, such as, e.g., SH-SY5Y cells, thatinclude a Clostridial toxin substrate comprising a SNAP-25 recognitionsequence; such as, e.g., a BoNT/A recognition sequence or a BoNT/Erecognition sequence; Neuro-2a cells, that include a Clostridial toxinsubstrate comprising a SNAP-25 recognition sequence; such as, e.g., aBoNT/A recognition sequence; and N1E-115 cells or SK-N-DZ cells, thatinclude a Clostridial toxin substrate comprising a SNAP-25 recognitionsequence; such as, e.g., a BoNT/E recognition sequence.

Neuronal hybrid cell lines, such as, e.g., murine, rat, primate andhuman hybrid neuronal cell lines can be useful in aspects of theinvention. Such hybrid cell lines include neuroblastoma/glioma hybrids,such as, e.g., N18 (ECACC 88112301), NG108-15 (ATCC HB-12317, ECACC88112302) and NG115-401 L (ECACC 87032003); neuroblastoma/motor neuronhybrids, such as, e.g., NSC-19 and NSC-34, which express motor neuroncharacteristics, display a multipolar neuron-like phenotype, expresshigh levels of choline acetyltransferase (CHAT), generate actionpotentials, express neurofilament triplet proteins and synthesize, storeand release acetylcholine, see, e.g., N. R. Cashman et al.,Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developingmotor neurons, 194(3) Dev. Dyn. 209-221 (1992); and Christopher J.Eggett et al., Development and characterisation of a glutamate-sensitivemotor neuronal cell line, 74(5) J. Neurochem. 1895-1902 (2000);neuroblastoma/dorsal root ganglion neuron hybrids, such as, e.g., F11,see, e.g., Doros Platika et al., Neuronal traits of clonal cell linesderived by fusion of dorsal root ganglia neurons with neuroblastomacells, 82(10) Proc. Natl. Acad. Sci. U.S.A. 3499-3503 (1985), ND-C(ECACC 92090913), ND-E (ECACC 92090915), ND-U1 (ECACC 92090916), ND3(ECACC 92090901), ND7/23 (ECACC 92090903), ND8/34 (ECACC 92090904),ND8/47, ND15 (ECACC 92090907), ND27 (ECACC 92090912);neuroblastoma/hippocampal neuron hybrids, such as, e.g., HN-33, see,e.g., Henry J. Lee et al., Neuronal properties and trophic activities ofimmortalized hippocampal cells from embryonic and young adult mice.10(6) J. Neurosci. 1779-1787 (1990). Thus, in an embodiment, a cellcapable of Clostridial toxin intoxication can be a hybrid neuron. Inaspects of this embodiment, a hybrid neuron can be, e.g., aneuroblastoma/glioma hybrid, a neuroblastoma/motor neuron hybrid, aneuroblastoma/root ganglion neuron hybrid and aneuroblastoma/hippocampal neuron hybrid. In further aspects of thisembodiment, a neuroblastoma/glioma hybrid can be, e.g., N18, NG108-15and NG115-401 L. In further aspects of this embodiment, aneuroblastoma/motor neuron hybrid can be, e.g., NSC-19 and NSC-32. Infurther aspects of this embodiment, a neuroblastoma/dorsal root ganglionneuron hybrid can be, e.g., ND8-47. In further aspects of thisembodiment, a neuroblastoma/root ganglion neuron hybrid can be, e.g.,F11, ND-C, ND-E, ND-U1, ND3, ND7/23, ND8/34, ND8/47, ND15 and ND27. Infurther aspects of this embodiment, a neuroblastoma/hippocampal neuronhybrid can be, e.g., HN-33.

The NG108-15 cell line is a hybrid of mouse neuroblastoma and rat gliomacells that binds BoNT/C1 at subnanomolar concentrations with an IC₅₀ of0.2 nM (0.18 ng of complex per microliter), reaching saturation at 6 nM,see, e.g., Noriko Yokosawa et al., Binding of Clostridium botulinum typeC neurotoxin to different neuroblastoma cell lines, 57(1) Infect. Immun.272-277 (1989); and Noriko Yokosawa et al., Binding of botulinum typeCl, D and E neurotoxins to neuronal cell lines and synaptosomes, 29(2)Toxicon 261-264 (1991). Based on binding data, the NG108-15 cell linemay contain both low and high affinity receptors for BoNT/C1. Asnon-limiting examples, cells useful for determining Clostridial toxinactivity according to a method disclosed in the present specificationcan include, a neuronal hybrid cell, such as, e.g., NG108-15 cells, thatinclude a Clostridial toxin substrate comprising a SNAP-25 recognitionsequence; such as, e.g., a BoNT/A recognition sequence, a BoNT/C1recognition sequence or a BoNT/E recognition sequence; and NG108-15cells, that include a Clostridial toxin substrate comprising a Syntaxinrecognition sequence; such as, e.g., a BoNT/C1 recognition sequence.

Spinal cord cell lines, such as, e.g., murine, rat, primate or humanspinal cord cell lines can be useful in aspects of the invention andinclude, without limitation, TE 189.T (ATCC CRL-7947) and M4b, see,e.g., Ana M. Cardenas et al., Establishment and characterization ofimmortalized neuronal cell lines derived from the spinal cord of normaland trisomy 16 fetal mice, an animal model of Down syndrome, 68(1) J.Neurosci. Res. 46-58 (2002). As an example, a human spinal cord cellline can be generated from precursors of human embryonic spinal cordcells (first trimester embryos) that are immortalized with atetracycline repressible v-myc oncogene as described in Ronghao Li etal., Motoneuron differentiation of immortalized human spinal cord celllines, 59(3) J. Neurosci. Res. 342-352 (2000). Such cells can beexpanded indefinitely in proliferative growth conditions before rapiddifferentiation (4-7 days) into functional neurons that express neuronalphenotypic markers such as choline acetyltransferase. As anotherexample, a murine spinal cord cell line can be prepared by immortalizingan embryonic spinal cord culture using transforming media. Such a spinalcord cell line can be, for example, the murine M4b line and can expressneuronal markers such as NSE, synaptophysin, MAP 2 and cholineacetyltransferase, and can release acetylcholine upon appropriatestimulation, see, e.g., Cardenas et al., supra, (2002). Thus, in anembodiment, a cell capable of Clostridial toxin intoxication can be aspinal cord cell. In aspects of this embodiment, a spinal cord cell canbe, e.g., TE 189.T and M4b.

Central nervous system (CNS) cell lines, such as, e.g., murine, rat,primate and human CNS cell lines, can be useful in aspects of theinvention. A useful CNS cell line can be, for example, a human CNS cellline immortalized with a tetracycline repressible v-myc oncogene asdescribed in Dinah W. Sah et al., Bipotent progenitor cell lines fromthe human CNS, 15(6) Nat. Biotechnol. 574-580 (1997). Upon repression ofthe oncogene, the cells differentiate into neurons. Thus, in anembodiment, a cell capable of Clostridial toxin intoxication can be aCNS cell.

Cerebral cortex cell lines, such as, e.g., murine, rat, primate andhuman cerebral cortex cell lines, can be useful in aspects of theinvention and include, without limitation, CNh, see, e.g., Ana M.Cardenas et al., Calcium signals in cell lines derived from the cerebralcortex of normal and trisomy 16 mice, 10(2) Neuroreport 363-369 (1999),HCN-1a (ATCC CRL-10442) and HCN-2 (ATCC CRL-10742). As an example,murine cortex primary cultures from 12-16 days embryos can beimmortalized, for example, by culturing the cells in conditioned mediafrom a rat thyroid cell line that induces transformation in vitro. Theimmortalized cells can be differentiated into neurons expressingneuronal markers using the appropriate media; these differentiated cellsexpress choline acetyltransferase and secrete acetylcholine andglutamate in response to depolarization and nicotine stimulation, see,e.g., David D. Allen et al., Impaired cholinergic function in cell linesderived from the cerebral cortex of normal and trisomy 16 mice, 12(9)Eur. J. Neurosci. 3259-3264 (2000). Thus, in an embodiment, a cellcapable of Clostridial toxin intoxication can be a cerebral cortex cell.In aspects of this embodiment, a cerebral cortex cell can be, e.g., CNh,HCN-1a and HCN-2.

Dorsal root ganglia cell lines, such as, e.g., murine, rat, primate andhuman dorsal root ganglia cell lines, can be useful in aspects of theinvention and include, without limitation, G4b, see, e.g., David D.Allen et al., A dorsal root ganglia cell line derived from trisomy 16fetal mice, a model for Down syndrome, 13(4) Neuroreport 491-496 (2002).Embryonic dorsal root ganglia primary cultures can be immortalized withtransforming conditioned media as described above. Upon differentiation,the cell line exhibits neuronal traits and lacks glial markers byimmunohistochemistry. Release of neurotransmitters such as acetylcholinecan be induced in response to potassium and nicotine, see, e.g., Allenet al., supra, (2002). Thus, in an embodiment, a cell capable ofClostridial toxin intoxication can be a dorsal root ganglia cell. Inaspects of this embodiment, a dorsal root ganglia cell can be, e.g.,G4b.

Hippocampal cell lines, such as, e.g., murine, rat, primate and humanhippocampal lines can be useful in aspects of the invention and include,without limitation, HT-4, see, e.g., K. Frederiksen et al.,Immortalization of precursor cells from the mammalian CNS, 1(6) Neuron439-448 (1988) and HT-22, see, e.g., John B. Davis and Pamela Maher,Protein kinase C activation inhibits glutamate-induced cytotoxicity in aneuronal cell line, 652(1) Brain Res. 169-173 (1994). As a non-limitingexample, the murine hippocampal cell line HT-22 can be useful in theinvention. As a further non-limiting example, the immortalized HN33hippocampal cell line can be useful in the invention. This hippocampalcell line was derived from the fusion of primary neurons from thehippocampus of postnatal day 21 mice with the N18TG2 neuroblastoma cellline, and, when differentiated, shares membrane properties with adulthippocampal neurons in primary culture, see, e.g., Henry J. Lee et al.,Neuronal Properties and Trophic Activities of Immortalized HippocampalCells from Embryonic and Young Adult Mice, 19(6) J. Neurosci. 1779-1787(1990); and Henry J. Lee et al., Immortalized young adult neurons fromthe septal region: generation and characterization, 52(1-2) Brain Res.Dev Brain Res. 219-228 (1990). Thus, in an embodiment, a cell capable ofClostridial toxin intoxication can be a hippocampal cell. In aspects ofthis embodiment, a hippocampal cell can be, e.g., HT-4, HT-22 and HN33.

A variety of non-neuronal cells are useful in aspects of the invention.Non-neuronal cells useful in aspects of the invention include, withoutlimitation, primary non-neuronal cells; immortalized or establishednon-neuronal cells; transformed non-neuronal cells; non-neuronal tumorcells; stably and transiently transfected non-neuronal cells and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman non-neuronal cells. Non-neuronal cells useful in aspects of theinvention further include, without limitation, any of the followingprimary or established cells: anterior pituitary cells; adrenal cells,such as. e.g., chromaffin cells of the adrenal medulla; pancreaticcells, such as. e.g., pancreatic acinar cells, pancreatic islet β cellsand insulinoma HIT or INS-1 cells; ovarian cells, such as. e.g.,steroid-producing ovarian cells; kidney cells, such as. e.g., innermedullary collecting duct (IMCD) cells; stomach cells, such as, e.g.,enterochromaffin cells; blood cells, such as. e.g., eurythrocytes,leucocytes, platelets, neutrophils, eosinophils, mast cells; epithelialcells, such as. e.g., those of the apical plasma membrane; fibroblasts;thyroid cells; chondrocytes; muscle cells; hepatocytes; glandular cellssuch as, e.g., pituitary cells, adrenal cells, chromaffin cells; andcells involved in glucose transporter (GLUT4) translocation. Thus, in anembodiment, a cell capable of Clostridial toxin intoxication can be anon-neuronal cell. In aspects of this embodiment, a non-neuronal cellcan be from a primary or established non-neuronal cell line from the,e.g., anterior pituitary cells, adrenal cells, pancreatic cells, ovariancells, kidney cells, stomach cells, blood cells, epithelial cells,fibroblasts, thyroid cells, chondrocytes, muscle cells, hepatocytes andglandular cells.

As non-limiting examples, cells useful for determining Clostridial toxinactivity according to a method disclosed in the present specificationcan include, a primary or established non-neuronal cell, such as, e.g.,chromaffin cells or pancreatic acinar cells, that include a Clostridialtoxin substrate comprising a SNAP-25 recognition sequence; such as,e.g., a BoNT/A recognition sequence or a BoNT/E recognition sequence; aprimary neuronal cell, such as, e.g., chromaffin cells or pancreaticacinar cells, that include a Clostridial toxin substrate comprising aVAMP recognition sequence; such as, e.g., a BoNT/B recognition sequenceor a TeNT recognition sequence; and a primary neuronal cell, such as,e.g., chromaffin cells or pancreatic acinar cells, that include aClostridial toxin substrate comprising a Syntaxin recognition sequence;such as, e.g., a BoNT/C1 recognition sequence.

As discussed above, cells useful in the invention include neuronal andnon-neuronal cells that express low or undetectable levels of endogenousreceptor but which have been transfected with, or otherwise engineeredto express, one or more exogenous nucleic acid molecules encoding one ormore Clostridial toxin receptors. The selection of the Clostridial toxinreceptor depends on which Clostridial toxin is to be assayed. As anon-limiting example, a neuronal or non-neuronal cell can be transientlyor stably engineered to express an exogenous nucleic acid moleculeencoding the fibroblast growth factor 3 receptor (FGFR3), which servesas a BoNT/A receptor, see, e.g., PCT Patent Application No. 2005/006421.As another non-limiting example, a neuronal or non-neuronal cell can betransiently or stably engineered to express an exogenous nucleic acidmolecule encoding a synaptic vesicle glycoprotein 2 (SV2) isoform, whichserves as a BoNT/A receptor, see, e.g., Min Dong et al., SV2 Is theProtein Receptor for Botulinum Neurotoxin A, Science (2006); S. Mahrholdet al, The Synaptic Vesicle Protein 2C Mediates the Uptake of BotulinumNeurotoxin A into Phrenic Nerves, 580(8) FEBS Lett. 2011-2014 (2006).Additionally, a neuronal or non-neuronal cell can be transiently orstably engineered to express multiple exogenous nucleic acid moleculesencoding FGFR3 and an SV2 isoform. As another non-limiting example, aneuronal or non-neuronal cell can be transiently or stably engineered toexpress an exogenous nucleic acid molecule encoding the synaptotagmin I,which serves as a BoNT/B receptor and as a BoNT/G receptor, see, e.g.,Min Dong et al., Synaptotagmins I and II mediate entry of botulinumneurotoxin B into cells, 162(7) J. Cell Biol. 1293-1303 (2003); andAndreas Rummel et al., Synaptotagmins I and II act as nerve cellreceptors for botulinum neurotoxin G, 279(29) J. Biol. Chem. 30865-30870(2004). As another non-limiting example, a neuronal or non-neuronal cellcan be transiently or stably engineered to express an exogenous nucleicacid molecule encoding the synaptotagmin II, which serves as a BoNT/Breceptor and as a BoNT/G receptor, see, e.g., Min Dong et al., supra,(2003); and Andreas Rummel et al., supra, (2004).

Thus in an embodiment, a neuronal or non-neuronal cell is transiently orstably engineered to express an exogenous nucleic acid molecule encodinga FGFR3. In aspects of this embodiment, a neuronal or non-neuronal cellis transiently or stably engineered to express an exogenous nucleic acidmolecule encoding the FGFR3 of SEQ ID NO: 173, the FGFR3 of SEQ ID NO:174 or the FGFR3 of SEQ ID NO: 175.

In another embodiment, a neuronal or non-neuronal cell is transiently orstably engineered to express an exogenous nucleic acid molecule encodinga SV2. In aspects of this embodiment, a neuronal or non-neuronal cell istransiently or stably engineered to express an exogenous nucleic acidmolecule encoding the SV2 of SEQ ID NO: 176, the SV2 of SEQ ID NO: 177,the SV2 of SEQ ID NO: 178 or the SV2 of SEQ ID NO: 179.

In another embodiment, a neuronal or non-neuronal cell is transiently orstably engineered to express an exogenous nucleic acid molecule encodinga FGFR3 and an exogenous nucleic acid molecule encoding a SV2. Inaspects of this embodiment, a neuronal or non-neuronal cell istransiently or stably engineered to express an exogenous nucleic acidmolecule encoding the FGFR3 of SEQ ID NO: 173, the FGFR3 of SEQ ID NO:174 or the FGFR3 of SEQ ID NO: 175 and an exogenous nucleic acidmolecule encoding the SV2 of SEQ ID NO: 176, the SV2 of SEQ ID NO: 177,the SV2 of SEQ ID NO: 178 or the SV2 of SEQ ID NO: 179.

In another embodiment, a neuronal or non-neuronal cell is transiently orstably engineered to express an exogenous nucleic acid molecule encodinga Synaptotagmin I. In aspects of this embodiment, a neuronal ornon-neuronal cell is transiently or stably engineered to express anexogenous nucleic acid molecule encoding the Synaptotagmin of SEQ ID NO:180.

In another embodiment, a neuronal or non-neuronal cell is transiently orstably engineered to express an exogenous nucleic acid molecule encodinga Synaptotagmin II. In aspects of this embodiment, a neuronal ornon-neuronal cell is transiently or stably engineered to express anexogenous nucleic acid molecule encoding the Synaptotagmin of SEQ ID NO:181.

Cells useful in aspects of the present invention further include,without limitation, transformed, tumor or other cells which over-expressone or more endogenous Clostridial toxin receptors or which express oneor more endogenous Clostridial toxin receptors. It is understood thatthe over-expressed receptor can be a wild type form of the receptor orcan include one or more amino acid modifications as compared to the wildtype receptor, with the proviso that the process of Clostridial toxinintoxication can still occur. As a non-limiting example, cells usefulfor determining BoNT/A activity encompass those which express orover-express a form of the fibroblast growth factor 3 receptor (FGFR3).As another non-limiting example, cells useful for determining BoNT/Bactivity encompass those which express or over-express a form ofsynaptotagmin I. As another non-limiting example, cells useful fordetermining BoNT/B activity encompass those which express orover-express a form of synaptotagmin II. As another non-limitingexample, cells useful for determining BoNT/G activity encompass thosewhich express or over-express a form of synaptotagmin I. As anothernon-limiting example, cells useful for determining BoNT/G activityencompass those which express or over-express a form of synaptotagminII.

Cells which express or over-express a form of the fibroblast growthfactor 3 receptor include, yet are not limited to, naturally occurringand genetically modified as well as primary and established myelomacells, bladder carcinoma cells, prostate carcinoma cells, thyroidcarcinoma cells and cervical carcinoma cells. Such cells useful inaspects of the invention further encompass, without limitation, humanmyeloma cell lines including H929 (ATCC CRL-9068; ECACC 95050415; DSMZACC 163), JIM-3, see, e.g., H. Barker et al., pp. 155-158 (J. Radl & B.van Camp eds., EURAGE Monoclonal Gammopathies III: Clinical Significanceand Basic Mechanisms, 1991), KMS-11, see, e.g., Masayoshi Namba et al.,Establishment of five human myeloma cell lines, 25(8) In Vitro Cell Dev.Biol. 723-729 (1989), KMS-18, see, e.g., Naozo Nakazawa et al.,Interphase detection of t(4;14)(p16.3;q32.3) by in situ hybridizationand FGFR3 over-expression in plasma cell malignancies, 117(2) CancerGenet. Cytogenet. 89-96 (2000), LB278, see, e.g., D. Ronchetti et al.,Characterization of the t(4;14)(p16.3;q32) in the KMS-18 multiplemyeloma cell line, 15(5) Leukemia 864-865 (2001), LB375, see, e.g.,Ronchetti et al., supra, (2001), LB1017, see, e.g., Ronchetti et al.,supra, (2001), LB2100, see, e.g., Ronchetti et al., supra, (2001), LP-1(DSMZ ACC 41), OPM-2 (DSMZ ACC 50), PCL1, see, e.g., Ronchetti et al.,supra, (2001), UTMC-2, see, e.g., Shuji Ozaki et al., Characterizationof a novel interleukin-6 autocrine-dependent human plasma cell line,8(12) Leukemia 2207-2213 (1994), which over-express FGFR3 due tochromosomal translocation t(4;14)(q16.3;q32.3) and other multiplemyeloma cells with a t(4:14) translocation; leukemia cells includingchronic myeloid leukemia (CML) cells such as CD34+ BCR-ABL+ cells; andbladder carcinoma cells including primary and other urothelial carcinomacells. One skilled in the art understands that these and other cellswhich over-express or express a form of the fibroblast growth factor 3receptor can be useful in determining BoNT/A activity according to amethod of the invention.

Thus, in an embodiment, a cell capable of Clostridial toxin intoxicationcan be a cell expressing an endogenous Clostridial toxin receptor. Inaspects of this embodiment, an endogenous Clostridial toxin receptorexpressed by a cell is a receptor for, e.g., BoNT/A, BoNT/B, BoNT/C1,BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT. In further aspects of thisembodiment, an endogenous Clostridial toxin receptor is, e.g., FGFR3,synaptotagmin I or synaptotagmin II. In another aspect of thisembodiment, a cell expressing an endogenous Clostridial toxin receptorcan be from, e.g., a primary myeloma cell line, an established myelomacell line, a primary bladder carcinoma cell line, an established bladdercarcinoma cell line, a primary cervical carcinoma cell line and anestablished cervical carcinoma cell line. In another embodiment, anFGFR3 expressing cell can be, e.g., a cell containing at(4;14)(q16.3;q32.3) chromosomal translocation. In further aspects ofthis embodiment, an FGFR3 expressing cell can be, e.g., H929, JIM-3,KMS-11, KMS-18, LB278, LB375, LB1017, LB2100, LP-1, OPM-2, PCL1 andUTMC-2. In further aspects of this embodiment, an FGFR3 expressing cellcan be, e.g., H929, JIM-3, KMS-11, KMS-18, LB278, LB375, LB1017, LB2100,LP-1, OPM-2, PCL1 and UTMC-2. As non-limiting examples, cells useful fordetermining Clostridial toxin activity according to a method disclosedin the present specification can include, an established myeloma cell,such as, e.g., KMS-11 or H929, that include a Clostridial toxinsubstrate comprising a SNAP-25 recognition sequence; such as, e.g., aBoNT/A recognition sequence; a primary or established bladder carcinomacell that includes a Clostridial toxin substrate comprising a SNAP-25recognition sequence; such as, e.g., a BoNT/A recognition sequence; anda primary or established cervical carcinoma cell that includes aClostridial toxin substrate comprising a SNAP-25 recognition sequence;such as, e.g., a BoNT/A recognition sequence.

Further such cells useful in aspects of the invention further encompass,without limitation, stably transfected cell lines expressing aClostridial toxin receptor. including, e.g., B9, see, e.g., Elizabeth E.Plowright et al., Ectopic expression of fibroblast growth factorreceptor 3 promotes myeloma cell proliferation and prevents apoptosis,95(3) Blood 992-998 (2000); TC, see, e.g., Hiroyuki Onose et al.,Over-expression of fibroblast growth factor receptor 3 in a humanthyroid carcinoma cell line results in overgrowth of the confluentcultures, 140(2) Eur. J. Endocrinol. 169-173 (1999); L6, see, e.g., M.Kana et al., Signal transduction pathway of human fibroblast growthfactor receptor 3. Identification of a novel 66-kDa phosphoprotein,272(10) J. Biol. Chem. 6621-6628 (1997); and CFK2, see, e.g., Janet E.Henderson et al., Expression of FGFR3 with the G380R achondroplasiamutation inhibits proliferation and maturation of CFK2 chondrocyticcells, 15(1) J. Bone Miner. Res. 155-165 (2000). One skilled in the artunderstands that these and other cells which over-express or express anactivated form of the fibroblast growth factor 3 receptor can be usefulin determining BoNT/A activity according to a method of the invention.Thus, in an embodiment, a cell capable of Clostridial toxin intoxicationcan be a cell stably expressing an exogenous Clostridial toxin receptor.In aspects of this embodiment, an exogenous Clostridial toxin receptorstably expressed by a cell is a receptor for, e.g., BoNT/A, BoNT/B,BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT. In further aspects ofthis embodiment, an exogenous Clostridial toxin receptor is, e.g.,FGFR3. In further aspects of this embodiment, an FGFR3 expressing cellcan be, e.g., B9, TC, L6 and CFK2. As non-limiting examples, cellsuseful for determining Clostridial toxin activity according to a methoddisclosed in the present specification can include a B9 cell whichstably express a nucleic acid molecule encoding a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate; a B9 cell which stablycontains a Clostridial toxin substrate, such as, e.g., a BoNT/Asubstrate; a TC cell which stably express a nucleic acid moleculeencoding a Clostridial toxin substrate, such as, e.g., a BoNT/Asubstrate; a TC cell which stably contains a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate; a L6 cell which stablyexpress a nucleic acid molecule encoding a Clostridial toxin substrate,such as, e.g., a BoNT/A substrate; a L6 cell which stably contains aClostridial toxin substrate, such as, e.g., a BoNT/A substrate; a CFK2cell which stably express a nucleic acid molecule encoding a Clostridialtoxin substrate, such as, e.g., a BoNT/A substrate; and a CFK2 cellwhich stably contains a Clostridial toxin substrate, such as, e.g., aBoNT/A substrate.

The cell compositions disclosed in the present specification include, inpart, a Clostridial toxin substrate. In is envisioned that any and allClostridial toxin substrate disclosed in the present specification canbe used. Thus, aspects of this embodiment include nucleic acidmolecules, such as, e.g., DNA and RNA, that encode a Clostridial toxinsubstrate disclosed in the present specification and peptide molecule orpeptidomimetic comprising a Clostridial toxin substrate disclosed in thepresent specification. Other aspects of this embodiment include, inpart, a Clostridial toxin recognition sequence including, withoutlimitation, a BoNT/A toxin recognition sequence, a BoNT/B toxinrecognition sequence, a BoNT/C1 toxin recognition sequence, a BoNT/Dtoxin recognition sequence, a BoNT/E toxin recognition sequence, aBoNT/F toxin recognition sequence, a BoNT/G toxin recognition sequenceand a TeNT toxin recognition sequence. Other aspects of this embodimentinclude, in part, a membrane targeting domain including, withoutlimitation, naturally occurring membrane targeting domains present inSNAP-25, naturally occurring SNAP-25 MTD variants, and non-naturallyoccurring SNAP-25 MTD variants, and SNAP-25 MTD peptidomimetics; andnaturally occurring membrane targeting domains present in syntaxin,naturally occurring syntaxin MTD variants, and non-naturally occurringsyntaxin MTD variants and syntaxin MTD peptidomimetics. Other aspects ofthis embodiment include, in part, a fluorescent protein including,without limitation, wild-type fluorescent proteins, naturally occurringvariants, genetically engineered variants, active peptide fragmentsderived from Aequorea fluorescent proteins, Anemonia fluorescentproteins, Anthozoa fluorescent proteins, Discosoma fluorescent proteins,Entacmeae fluorescent proteins, Heteractis fluorescent proteins,Montastrea fluorescent proteins, Renilla fluorescent proteins, Zoanthusfluorescent proteins and fluorescent binding proteins. Non-limitingexamples of fluorescent proteins include, e.g., EBFP, ECFP, AmCyan,AcGFP, ZsGreen, Vitality® hrGFP, EGFP, Monster Green® hMGFP, EYFP,ZsYellow, DsRed-Express, DsRed2, DsRed, AsRed2 and HcRed1. Non-limitingexamples of fluorescent binding proteins include, e.g., a tetracysteinepeptide, an AGT and a dehalogenase.

The cell compositions disclosed in the present specification include, inpart, a cell that transiently contains a Clostridial toxin substrate. Asused herein, the term “transiently containing” means a Clostridial toxinsubstrate that is temporarily introduced into a cell in order to performthe assays disclosed in the present specification. By definition, inorder to perform the assays disclosed in the present specification atleast 50% of the cells comprising a cell population must contain anexogenous Clostridial toxin substrate. As used herein, the term “cellpopulation” means the total number of cells used in a method thattransiently introduces a Clostridial toxin substrate for a given assay.As a non-limiting example, given a cell population comprising 1.5×10⁵cells, at least 7.5×10⁴ cells must contain a non-naturally occurringClostridal toxin substrate after transduction using, e.g., an adenoviralmethod or a lentiviral method. As another non-limiting example, given acell population comprising 1.5×10⁵ cells, at least 7.5×10⁴ cells mustcontain an exogenous Clostridal toxin substrate after transfectionusing, e.g., a protein transfection method. Thus, aspects of a celltransiently containing a Clostridial toxin substrate disclosed in thespecification may include a cell that contains a substrate for, e.g., atmost about one day, at most about two days, at most about three days, atmost about four days, at most about five days, and at most about sixdays, at most about seven days, at most about eight days, at most aboutnine days and at most about ten days and wherein the cell populationcontaining a Clostridial toxin substrate comprises, e.g., at least 50%of the cells within the cell population, at least 60% of the cellswithin the cell population, at least 70% of the cells within the cellpopulation, at least 80% of the cells within the cell population, and atleast 90% of the cells within the cell population.

Thus, in an embodiment, a composition comprises a cell that transientlycontains a nucleic acid molecule that encodes an exogenous Clostridialtoxin substrate capable of being localized to the plasma membrane ofsaid cell wherein said cell is capable of Clostridial toxinintoxication, wherein said substrate comprises a fluorescent member, amembrane targeting domain and a Clostridial toxin recognition sequencecomprising a cleavage site, where the cleavage site intervenes betweensaid fluorescent member and said membrane localization domain.

In another embodiment, a composition comprises a cell population, saidcell population comprising cells that transiently contains a nucleicacid molecule that encodes an exogenous Clostridial toxin substratecapable of being localized to the plasma membrane of said cells whereinsaid cells are capable of Clostridial toxin intoxication, wherein saidsubstrate comprises a fluorescent member, a membrane targeting domainand a Clostridial toxin recognition sequence comprising a cleavage site,where the cleavage site intervenes between said fluorescent member andsaid membrane localization domain and wherein greater than 50% of saidcell population comprises said cells containing said exogenousClostridial toxin substrate. In aspects of this embodiment, theClostridial toxin substrate capable of being localized to the plasmamembrane encoded by the nucleic acid molecule can be, e.g., a BoNT/Asubstrate, a BoNT/B substrate, a BoNT/C1 substrate, a BoNT/D substrate,a BoNT/E substrate, a BoNT/F substrate, a BoNT/G substrate or a TeNTsubstrate. As non-limiting examples, cells useful for determiningClostridial toxin activity according to a method disclosed in thepresent specification can include SH-SY5Y cells such as, e.g.,differentiated SH-SY5Y cells and SH-SY5Y cells which transiently expressa nucleic acid molecule encoding a Clostridial toxin substrate, such as,e.g., a BoNT/A substrate or a BoNT/E substrate; NG108-15 cells such as,e.g., differentiated NG108-15 cells and NG108-15 cells which transientlyexpress a nucleic acid molecule encoding a Clostridial toxin substrate,such as, e.g., a BoNT/A substrate or a BoNT/E substrate; Neuro-2A cellssuch as, e.g., differentiated Neuro-2A cells and Neuro-2A cells whichtransiently express a nucleic acid molecule encoding a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate; N1E-115 cells such as,e.g., differentiated N1E-115 cells and N1E-115 cells which transientlyexpress a nucleic acid molecule encoding a Clostridial toxin substrate,such as, e.g., a BoNT/E substrate; and SK-N-DZ cells such as, e.g.,differentiated SK-N-DZ cells and SK-N-DZ cells which transiently expressa nucleic acid molecule encoding a Clostridial toxin substrate, such as,e.g., a BoNT/E substrate.

In another embodiment, a composition comprises a cell that transientlycontains an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of said cell wherein said cell iscapable of Clostridial toxin intoxication, wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain.

In another embodiment, a composition comprises a cell population, saidcell population comprising cells that transiently contains an exogenousClostridial toxin substrate capable of being localized to the plasmamembrane of said cells wherein said cells are capable of Clostridialtoxin intoxication, wherein said substrate comprises a fluorescentmember, a membrane targeting domain and a Clostridial toxin recognitionsequence comprising a cleavage site, where the cleavage site intervenesbetween said fluorescent member and said membrane localization domainand wherein greater than 50% of said cell population comprises saidcells containing said exogenous Clostridial toxin substrate. In aspectsof this embodiment, the Clostridial toxin substrate capable of beinglocalized to the plasma membrane can be, e.g., a BoNT/A substrate, aBoNT/B substrate, a BoNT/C1 substrate, a BoNT/D substrate, a BoNT/Esubstrate, a BoNT/F substrate, a BoNT/G substrate or a TeNT substrate.As non-limiting examples, cells useful for determining Clostridial toxinactivity according to a method disclosed in the present specificationcan include SH-SY5Y cells such as, e.g., differentiated SH-SY5Y cellsand SH-SY5Y cells which transiently contain a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate or a BoNT/E substrate;NG108-15 cells such as, e.g., differentiated NG108-15 cells and NG108-15cells which transiently contain a Clostridial toxin substrate, such as,e.g., a BoNT/A substrate or a BoNT/E substrate; Neuro-2A cells such as,e.g., differentiated Neuro-2A cells and Neuro-2A cells which transientlycontain a Clostridial toxin substrate, such as, e.g., a BoNT/Asubstrate; N1E-115 cells such as, e.g., differentiated N1E-115 cells andN1E-115 cells which transiently contain a Clostridial toxin substrate,such as, e.g., a BoNT/E substrate; and SK-N-DZ cells such as, e.g.,differentiated SK-N-DZ cells and SK-N-DZ cells which transiently containa Clostridial toxin substrate, such as, e.g., a BoNT/E substrate.

The cell compositions disclosed in the present specification include, inpart, a cell that stably contains a Clostridial toxin substrate. As usedherein, the term “stably containing” means a Clostridial toxin substratethat is introduced into a cell and maintained for long periods of timein order to perform the fluorescence assays of the present invention.Stably-maintained nucleic acid molecules encompass stably-maintainednucleic acid molecules that are extra-chromosomal and replicateautonomously and stably-maintained nucleic acid molecules that areintegrated into the chromosomal material of the cell and replicatenon-autonomously. Thus aspects of a cell stably containing a Clostridialtoxin substrate disclosed in the specification may include a cell thatcontains a substrate for, e.g., at least ten days, at least 20 two days,at least 30 days, at least forty days, at least 50 days, and at least 60days, at least 70 days, at least 80 days, at least 90 days and at least100 days. Other aspects of a cell stably containing a Clostridial toxinsubstrate disclosed in the specification may include a cell thatcontains a substrate for, e.g., at least 100 days, at least 200 days, atleast 300 days, at least 400 days, and at least 500 days. Still otheraspects of a cell stably containing a Clostridial toxin substratedisclosed in the specification may include a cell that permanentlycontains a Clostridial toxin substrate.

Thus, in an embodiment, a composition comprises a cell stably containinga nucleic acid molecule that encodes an exogenous Clostridial toxinsubstrate capable of being localized to the plasma membrane of said cellwherein said cell is capable of Clostridial toxin intoxication, andwherein said substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between saidfluorescent member and said membrane localization domain.

In another embodiment, a composition comprises a cell population, saidcell population comprising cells stably containing a nucleic acidmolecule that encodes an exogenous Clostridial toxin substrate capableof being localized to the plasma membrane of said cells wherein saidcells are capable of Clostridial toxin intoxication, and wherein saidsubstrate comprises a fluorescent member, a membrane targeting domainand a Clostridial toxin recognition sequence comprising a cleavage site,where the cleavage site intervenes between said fluorescent member andsaid membrane localization domain. In aspects of this embodiment, theClostridial toxin substrate capable of being localized to the plasmamembrane encoded by the nucleic acid molecule can be, e.g., a BoNT/Asubstrate, a BoNT/B substrate, a BoNT/C1 substrate, a BoNT/D substrate,a BoNT/E substrate, a BoNT/F substrate, a BoNT/G substrate or a TeNTsubstrate. As non-limiting examples, cells useful for determiningClostridial toxin activity according to a method disclosed in thepresent specification can include SH-SY5Y cells such as, e.g.,differentiated SH-SY5Y cells and SH-SY5Y cells which stably express anucleic acid molecule encoding a Clostridial toxin substrate, such as,e.g., a BoNT/A substrate or a BoNT/E substrate; NG108-15 cells such as,e.g., differentiated NG108-15 cells and NG108-15 cells which stablyexpress a nucleic acid molecule encoding a Clostridial toxin substrate,such as, e.g., a BoNT/A substrate or a BoNT/E substrate; Neuro-2A cellssuch as, e.g., differentiated Neuro-2A cells and Neuro-2A cells whichstably express a nucleic acid molecule encoding a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate; KMS-11 cells such as,e.g., differentiated KMS-11 cells and KMS-11 cells which stably expressa nucleic acid molecule encoding a Clostridial toxin substrate, such as,e.g., a BoNT/A substrate; N1E-115 cells such as, e.g., differentiatedN1E-115 cells and N1E-115 cells which stably express a nucleic acidmolecule encoding a Clostridial toxin substrate, such as, e.g., a BoNT/Esubstrate; and SK-N-DZ cells such as, e.g., differentiated SK-N-DZ cellsand SK-N-DZ cells which stably express a nucleic acid molecule encodinga Clostridial toxin substrate, such as, e.g., a BoNT/E substrate.

In another embodiment, a composition comprises a cell stably containingan exogenous Clostridial toxin substrate capable of being localized tothe plasma membrane of said cell wherein said cell is capable ofClostridial toxin intoxication, and wherein said substrate comprises afluorescent protein, a membrane targeting domain and a Clostridial toxinrecognition sequence comprising a cleavage site, where the cleavage siteintervenes between said fluorescent protein and said membranelocalization domain.

In another embodiment, a composition comprises a cell population, saidcell population comprising cells stably containing an exogenousClostridial toxin substrate capable of being localized to the plasmamembrane of said cells wherein said cells are capable of Clostridialtoxin intoxication, and wherein said substrate comprises a fluorescentmember, a membrane targeting domain and a Clostridial toxin recognitionsequence comprising a cleavage site, where the cleavage site intervenesbetween said fluorescent member and said membrane localization domain.In aspects of this embodiment, the Clostridial toxin substrate capableof being localized to the plasma membrane can be, e.g., a BoNT/Asubstrate, a BoNT/B substrate, a BoNT/C1 substrate, a BoNT/D substrate,a BoNT/E substrate, a BoNT/F substrate, a BoNT/G substrate or a TeNTsubstrate. As non-limiting examples, cells useful for determiningClostridial toxin activity according to a method disclosed in thepresent specification can include SH-SY5Y cells such as, e.g.,differentiated SH-SY5Y cells and SH-SY5Y cells which stably contain aClostridial toxin substrate, such as, e.g., a BoNT/A substrate or aBoNT/E substrate; NG108-15 cells such as, e.g., differentiated NG108-15cells and NG108-15 cells which stably contain a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate or a BoNT/E substrate;Neuro-2A cells such as, e.g., differentiated Neuro-2A cells and Neuro-2Acells which stably contain a Clostridial toxin substrate, such as, e.g.,a BoNT/A substrate; KMS-11 cells such as, e.g., differentiated KMS-11cells and KMS-11 cells which stably contain a Clostridial toxinsubstrate, such as, e.g., a BoNT/A substrate; N1E-115 cells such as,e.g., differentiated N1E-115 cells and N1E-115 cells which stablycontain a Clostridial toxin substrate, such as, e.g., a BoNT/Esubstrate; and SK-N-DZ cells such as, e.g., differentiated SK-N-DZ cellsand SK-N-DZ cells which stably contain a Clostridial toxin substrate,such as, e.g., a BoNT/E substrate.

As mentioned above, a nucleic acid molecule can be used to express aClostridial toxin substrate disclosed in the present specification. Itis envisioned that any and all methods for introducing a nucleic acidmolecule into a cell can be used. Methods useful for introducing anucleic acid molecule into a cell including, without limitation, calciumphosphate-mediated, DEAE dextran-mediated, lipid-mediated,polybrene-mediated, polylysine-mediated, viral-mediated, microinjection,protoplast fusion, biolistic, electroporation and conjugation to anantibody, gramacidin S, artificial viral envelope or other intracellularcarrier such as TAT, see, e.g., Introducing Cloned Genes into CulturedMammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., MolecularCloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001); Alessia Colosimoet al., Transfer and expression of foreign genes in mammalian cells,29(2) Biotechniques 314-318, 320-322, 324 (2000); Philip Washbourne & A.Kimberley McAllister, Techniques for gene transfer into neurons, 12(5)Curr. Opin. Neurobiol. 566-573 (2002); and Current Protocols inMolecular Biology, John Wiley and Sons, pp 9.16.4-9.16.11 (2000). Oneskilled in the art understands that selection of a specific method tointroduce a nucleic acid molecule into a cell will depend, in part, onwhether the cell will transiently contain the Clostridial toxinsubstrate or whether the cell will stably contain the Clostridial toxinsubstrate.

In an aspect of this embodiment, a chemical-mediated method, termedtransfection, is used to introduce a nucleic acid molecule expressing aClostridial toxin substrate into a cell. In chemical-mediated methods oftransfection the chemical reagent forms a complex with the nucleic acidthat facilitates its uptake into the cells. Such chemical reagentsinclude, without limitation, calcium phosphate-mediated, see, e.g.,Martin Jordan & Florian Worm, Transfection of adherent and suspendedcells by calcium phosphate, 33(2) Methods 136-143 (2004);diethyl-laminoethyl (DEAE) dextran-mediated, lipid-mediated, cationicpolymer-mediated like polyethyleneimine (PEI)-mediated andpolylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang etal., Polyethylenimine strategies for plasmid delivery to brain-derivedcells, 33(2) Methods 144-150 (2004). Such chemical-mediated deliverysystems can be prepared by standard methods and are commerciallyavailable, see, e.g., CellPhect Transfection Kit (Amersham Biosciences,Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate andDEAE Dextran, (Stratagene, Inc., La Jolla, Calif.); Lipofectamine™Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); ExGen 500Transfection kit (Fermentas, Inc., Hanover, Md.), and SuperFect andEffectene Transfection Kits (Qiagen, Inc., Valencia, Calif.).

In another aspect of this embodiment, a physical-mediated method is usedto introduce a nucleic acid molecule expressing a Clostridial toxinsubstrate into a cell. Physical reagents include, without limitation,electroporation, biolistic and microinjection. Biolistics andmicroinjection techniques perforate the cell wall in order to introducethe nucleic acid molecule into the cell, see, e.g., Jeike E. Biewenga etal., Plasmid-mediated gene transfer in neurons using the biolisticstechnique, 71(1) J. Neurosci. Methods. 67-75 (1997); and John O'Brien &Sarah C. R. Lummis, Biolistic and diolistic transfection: using the genegun to deliver DNA and lipophilic dyes into mammalian cells, 33(2)Methods 121-125 (2004). Electroporation, also termedelectropermeabilization, uses brief, high-voltage, electrical pulses tocreate transient pores in the membrane through which the nucleic acidmolecules enter and be used effectively for stable and transienttransfections of all cell types, see, e.g., M. Golzio et al., In vitroand in vivo electric field-mediated permeabilization, gene transfer, andexpression, 33(2) Methods 126-135 (2004); and Oliver Greschet al., Newnon-viral method for gene transfer into primary cells, 33(2) Methods151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termedtransduction, is used to introduce a nucleic acid molecule expressing aClostridial toxin substrate into a cell. In viral-mediated methods oftransient transduction, the process by which viral particles infect andreplicate in a host cell has been manipulated in order to use thismechanism to introduce a nucleic acid molecule into the cell.Viral-mediated methods have been developed from a wide variety ofviruses including, without limitation, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex viruses, picornaviruses,alphaviruses and baculoviruses, see, e.g., Armin Blesch, Lentiviral andMLV based retroviral vectors for ex vivo and in vivo gene transfer,33(2) Methods 164-172 (2004); and Maurizio Federico, From lentivirusesto lentivirus vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.Poeschla, Non-primate lentiviral vectors, 5(5) Curr. Opin. Mol. Ther.529-540 (2003); Karim Benihoud et al, Adenovirus vectors for genedelivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H. Bueler,Adeno-associated viral vectors for gene transfer and gene therapy,380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al., Adenovirus andadeno-associated virus vectors, 21(12) DNA Cell Biol. 895-913 (2002);Edward A. Burton et al., Gene delivery using herpes simplex virusvectors, 21(12) DNA Cell Biol. 915-936 (2002); Paola Grandi et al.,Targeting HSV amplicon vectors, 33(2) Methods 179-186 (2004); IlyaFrolov et al., Alphavirus-based expression vectors: strategies andapplications, 93(21) Proc. Natl. Acad. Sci. U.S.A. 11371-11377 (1996);Markus U. Ehrengruber, Alphaviral gene transfer in neurobiology, 59(1)Brain Res. Bull. 13-22 (2002); Thomas A. Kost & J. Patrick Condreay,Recombinant baculoviruses as mammalian cell gene-delivery vectors, 20(4)Trends Biotechnol. 173-180 (2002); and A. Huser & C. Hofmann,Baculovirus vectors: novel mammalian cell gene-delivery vehicles andtheir applications, 3(1) Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, areoften selected for mammalian cell transduction because adenoviruseshandle relatively large nucleic acid molecules of about 36 kd, areproduced at high titer, and can efficiently infect a wide variety ofboth dividing and non-dividing cells, see, e.g., Wim T. J. M. C. Hermenset al., Transient gene transfer to neurons and glia: analysis ofadenoviral vector performance in the CNS and PNS, 71(1) J. Neurosci.Methods 85-98 (1997); and Hiroyuki Mizuguchi et al., Approaches forgenerating recombinant adenovirus vectors, 52(3) Adv. Drug Deliv. Rev.165-176 (2001). Transduction using adenoviral-based system do notsupport prolonged protein expression because the nucleic acid moleculeis carried from an episome in the cell nucleus, rather than beingintegrated into the host cell chromosome. Adenovirual vector systems andspecific protocols for how to use such vectors are disclosed in, e.g.,ViraPower™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Adenoviral Expression System Instruction Manual25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002); and AdEasy™Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif.) andAdEasy™ Adenoviral Vector System Instruction Manual 064004f, Stratagene,Inc.

Nucleic acid molecule delivery can also use single-stranded RNAretroviruses viruses, such as, e.g., oncoretroviruses and lentiviruses.Retroviral-mediated transduction often produce transduction efficienciesclose to 100%, can easily control the proviral copy number by varyingthe multiplicity of infection (MOI), and can be used to eithertransiently or stably transduce cells, see, e.g., Tiziana Tonini et al.,Transient production of retroviral- and lentiviral-based vectors for thetransduction of Mammalian cells, 285 Methods Mol. Biol. 141-148 (2004);Armin Blesch, Lentiviral and MLV based retroviral vectors for ex vivoand in vivo gene transfer, 33(2) Methods 164-172 (2004); FélixRecillas-Targa, Gene transfer and expression in mammalian cell lines andtransgenic animals, 267 Methods Mol. Biol. 417-433 (2004); and RolandWolkowicz et al., Lentiviral vectors for the delivery of DNA intomammalian cells, 246 Methods Mol. Biol. 391-411 (2004). Retroviralparticles consist of an RNA genome packaged in a protein capsid,surrounded by a lipid envelope. The retrovirus infects a host cell byinjecting its RNA into the cytoplasm along with the reversetranscriptase enzyme. The RNA template is then reverse transcribed intoa linear, double stranded cDNA that replicates itself by integratinginto the host cell genome. Viral particles are spread both vertically(from parent cell to daughter cells via the provirus) as well ashorizontally (from cell to cell via virions). This replication strategyenables long-term persist expression since the nucleic acid molecules ofinterest are stably integrated into a chromosome of the host cell,thereby enabling long-term expression of the protein. For instance,animal studies have shown that lentiviral vectors injected into avariety of tissues produced sustained protein expression for more than 1year, see, e.g., Luigi Naldini et al., In vivo gene delivery and stabletransduction of non-dividing cells by a lentiviral vector, 272(5259)Science 263-267 (1996). The Oncoretroviruses-derived vector systems,such as, e.g., Moloney murine leukemia virus (MoMLV), are widely usedand infect many different non-dividing cells. Lentiviruses can alsoinfect many different cell types, including dividing and non-dividingcells and possess complex envelope proteins, which allows for highlyspecific cellular targeting.

Retroviral vector systems and specific protocols for how to use suchvectors are disclosed in, e.g., U.S. patent Nos. Manfred Gossen &Hermann Bujard, Tight control of gene expression in eukaryotic cells bytetracycline-responsive promoters, U.S. Pat. No. 5,464,758 (Nov. 7,1995) and Hermann Bujard & Manfred Gossen, Methods for regulating geneexpression, U.S. Pat. No. 5,814,618 (Sep. 29, 1998) David S. Hogness,Polynucleotides encoding insect steroid hormone receptor polypeptidesand cells transformed with same, U.S. Pat. No. 5,514,578 (May 7, 1996)and David S. Hogness, Polynucleotide encoding insect ecdysone receptor,U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,Progesterone receptor having C. terminal hormone binding domaintruncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta Vegetoet al., Mutated steroid hormone receptors, methods for their use andmolecular switch for gene therapy, U.S. Pat. No. 5,874,534 (Feb. 23,1999) and Elisabetta Vegeto et al., Mutated steroid hormone receptors,methods for their use and molecular switch for gene therapy, U.S. Pat.No. 5,935,934 (Aug. 10, 1999). Furthermore, such viral delivery systemscan be prepared by standard methods and are commercially available, see,e.g., BD™ Tet-Off and Tet-On Gene Expression Systems (BDBiosciences-Clonetech, Palo Alto, Calif.) and BD™ Tet-Off and Tet-OnGene Expression Systems User Manual, PT3001-1, BD Biosciences Clonetech,(Mar. 14, 2003), GeneSwitch™ System (Invitrogen, Inc., Carlsbad, Calif.)and GeneSwitch™ System A Mifepristone-Regulated Expression System forMammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002);ViraPower™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad,Calif.) and ViraPower™ Lentiviral Expression System Instruction Manual25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003); and CompleteControl® Retroviral Inducible Mammalian Expression System (Stratagene,La Jolla, Calif.) and Complete Control® Retroviral Inducible MammalianExpression System Instruction Manual, 064005e.

As mentioned above, a Clostridial toxin substrate disclosed in thepresent specification can be introduced into a cell. It is envisionedthat any and all methods using a delivery agent to introduce aClostridial toxin substrate into a cell population can be used. As usedherein, the term “delivery agent” means any molecule that enables orenhances internalization of a covalently-linked, non-covalently-linkedor in any other manner associated with a Clostridial toxin substrateinto a cell. Thus, the term “delivery agent” encompasses, withoutlimitation, proteins, peptides, peptidomimetics, small molecules,nucleic acid molecules, liposomes, lipids, viruses, retroviruses andcells that, without limitation, transport a covalently or non-covalentlylinked substrate to the cell membrane, cell cytoplasm or nucleus. Itfurther is understood that the term “delivery agent” encompassesmolecules that are internalized by any mechanism, including deliveryagents which function via receptor mediated endocytosis and those whichare independent of receptor mediated endocytosis.

It is also envisioned that any and all methods useful for introducing aClostridial toxin substrate with a delivery agent into a cell populationcan be useful with the proviso that this method introduce a Clostridialtoxin substrate disclosed in the present specification in at least 50%of the cells within a given cell population. Thus, aspects of thisembodiment can include a cell population in which, e.g., at least 90% ofthe given cell population contains a Clostridial toxin substrate, atleast 80% of the given cell population contains a Clostridial toxinsubstrate, at least 70% of the given cell population contains aClostridial toxin substrate, at least 60% of the given cell populationcontains a Clostridial toxin substrate, at least 50% of the given cellpopulation contains a Clostridial toxin substrate.

It is also envisioned that any and all methods useful for introducing aClostridial toxin substrate disclosed in the present specificationlinked to a delivery agent can be useful, including methods thatcovalently link the delivery agent to the substrate and methods thatnon-covalently link the delivery agent to the substrate. Covalentlinking methods that attach a delivery agent to a Clostridial toxinsubstrate can include chemical conjugation and genetically producedfusion proteins. In one non-limiting method, a polynucleotide, such as,e.g., a plasmid or oligonucleotide, is attached to a Clostridial toxinsubstrate by conjugation chemistry and introduced into the cell using amethod useful for introducing a nucleic acid molecule into a cellpopulation as described in the present specification. In anothernon-limiting method, a lipid, such as, e.g., a cationic liposome, isattached to a Clostridial toxin substrate by conjugation chemistry andintroduced into the cell using a method useful for introducing a nucleicacid molecule into a cell population as described in the presentspecification. In yet another non-limiting method, a peptide, isattached to a Clostridial toxin substrate by conjugation chemistry andintroduced into the cell using a protein delivery method describedbelow. In yet another non-limiting method, a peptide is attached to aClostridial toxin substrate by producing a nucleic acid molecule thatencodes the peptide delivery agent and substrate as an operationallylinked fusion protein and this fusion protein is introduced into thecell using a protein delivery method described below.

In an aspect of the present invention, a Clostridial toxin substratedisclosed in the present specification can be introduced into a cellusing a peptide delivery agent to produce a cell transiently containinga Clostridial toxin substrate capable of being localized to the plasma.It is envisioned that a variety of peptide delivery agents can becovalently linked to a Clostridial toxin substrate, including, withoutlimitation, the active fragment of protein transduction peptides; theactive fragment of cell permeant peptides; phosphopeptides; the activefragment of membrane-translocating peptides; the active fragment ofsecreted proteins; the active fragment of nuclear localization signalpeptides; predominantly hydrophobic peptides; predominantly α-helicalpeptides, such as, e.g., amphipathic-helical peptide; predominantlybasic peptides, such as, e.g., basic amphipathic peptides; peptidescontaining D-amino acids; short peptides, such as, e.g., KDEL; denaturedpeptides linked to a denatured or folded Clostridial toxin substrate asdescribed in, e.g., Steven F. Dowdy, Methods for Transducing FusionMolecules, PCT Publication No. WO99/55899 (Nov. 11, 1999); and Steven F.Dowdy, Novel Transduction Molecules and Methods for Using the Same, PCTPublication No. WO0/62067 (Oct. 19, 2000); and, and any other denaturedor folded, modified or unmodified, naturally occurring or syntheticpeptide, peptidomimetics or analogs thereof.

It is envisioned that any and all peptide lengths of a delivery agentcan be useful in aspects of the present invention. In aspects of thisembodiment, therefore, a delivery agent useful for introducing aClostridial toxin substrate disclosed in the present specification in acell can be a peptide or peptidomimetic having a length of less than 10residues, a length of less than 20 residues, a length of less than 30residues, a length of less than 40 residues, or a length of less than 50residues.

As non-limiting examples, delivery agents suitable for introducing aClostridial toxin substrate disclosed in the present specification intoa cell include polylysine; ciliary neurotrophic factor (CNTF) or anactive fragment thereof; caveolin or an active fragment thereof;interleukin-1 (IL-1) or an active fragment thereof; thioredoxin or anactive fragment thereof; homeodomain-derived peptides or an activefragment thereof, such as, e.g., Antennapedia (Antp) or an activefragment thereof, like penetratin-1 (SEQ ID NO: 160), Engrailed 1 (En1)or an active fragment thereof, Engrailed 2 (En2) or an active fragmentthereof, Hoxb-4 or an active fragment thereof, Hoxa-5 or an activefragment thereof, Hoxc-8 or an active fragment thereof, and Knotted-1(KN1) or an active fragment thereof; fibroblast growth factor-1 (FGF-1)or an active fragment thereof; Kaposi fibroblast growth factor (kFGF) oran active fragment thereof, such as, e.g., AAVALLPAVLLALLAP (SEQ ID NO:169); human 3 integrin or an active fragment thereof such as, e.g., ahydrophobic signal sequence; a nuclear localization sequence (NLS) or anactive fragment thereof, such as, e.g., TPPKKKRKVEDP (SEQ ID NO: 170);FGF-2 or an active fragment thereof; transportan or an active fragmentthereof, such as, e.g., GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 171);lactoferrin or an active fragment thereof; VP22 or an active fragmentthereof; HIV type I transactivator (HIV TAT) or an active fragmentthereof, such as, e.g., (YGRKKRRQRRR; SEQ ID NO: 168); or a heat shockprotein such as HSP70 or an active fragment thereof. These andadditional delivery agents are well known in the art as described in,e.g., Steven R. Schwarze and Steven F. Dowdy, In vivo proteintransduction: intracellular delivery of biologically active proteins,compounds and DNA, 21(2) Trends Pharmacol. Sci. 45-48 (2000); Dara J.Dunican and Patrick Doherty, Designing cell-permeant phosphopeptides tomodulate intracellular signaling pathways, 60(1) Biopolymers 45-60(2001); K. G.

Ford et al., Protein transduction: an alternative to geneticintervention? 8(1) Gene Ther. 1-4 (2001); Alain Prochiantz, Messengerproteins: homeoproteins, TAT and others, 12(4) Curr. Opin. Cell Biol.400-406 (2000); J. J. Schwartz and S. Zhang, Peptide-mediated cellulardelivery, 2(2) Curr. Opin. Mol. Ther. 162-167 (2000); and Steven F.Dowdy, Protein Transduction System and Methods of Use Thereof, PCTPublication No. WO00/34308 (Jun. 15, 2000).

In an embodiment, a delivery agent can be a homeoprotein or an activefragment thereof, such as, e.g., a homeodomain or an active fragmentthereof. Homeoproteins are helix-turn-helix proteins that contain aDNA-binding domain of about 60 residues, denoted the homeodomain. Avariety of homeoproteins, homeodomains and active fragments thereof canbe delivery agents useful in the invention including, withoutlimitation, Antennapedia, Engrailed1 (En1), Engrailed2 (En2), Hoxa-5,Hoxc-8, Hoxb-4 and Knotted-1 (KN1). As an example, En1 and En1 have beenexpressed in COS-7 cells, where they are first secreted and theninternalized by other cells, see, e.g., Prochiantz, supra, (2000).Delivery agents using peptides derived from homeodomains and methods ofusing such agents are described in, e.g., Gérard Chassaing & AlainProchiantz, Peptides which can be Used as Vectors for the IntracellularAddresing of Active Molecules, U.S. Pat. No. 6,080,724 (Jun. 27, 2000).

In an aspect of this embodiment, a substrate composition of theinvention includes a delivery agent which is the homeodomain protein,Antennapedia, or an active fragment thereof. Antennapedia is a member ofa family of developmentally important Drosophila homeoproteins whichtranslocate across neuronal membranes. The third helix of theAntennapedia homeodomain, the 16 residue peptide “penetratin-1” (SEQ IDNO: 160), is internalized into live cells. The internalization occursboth at 37° C. and 4° C., indicating that delivery is neitherreceptor-mediated nor energy-dependent. Additional delivery agentsinclude peptides and peptidomimetics related in sequence to Penetratin-1such as, without limitation, one of the peptides shown below in Table 11or another penetratin-derived peptide or peptidomimetic, including aretroinverse or all D-amino acid peptide or peptidomimetic, or a relatedbut non-α-helical peptide or peptidomimetic, see, e.g., Chassaing &Prochiantz, supra, (2000). In one embodiment, such a penetratin-derivedpeptide retains the tryptophan, phenylalanine and glutamine residues ofpenetratin-1 (SEQ ID NO: 160).

TABLE 11 Penetratin-Derived Peptides Useful As Delivery Agents PeptideSequence SEQ ID NO: 43-58 RQIKIWFQNRRMKWKK 160 58-43 KKWKMRRNQFWIKIQR161 43-58 RQIKIWFQNRRMKWKK 162 Pro50 RQIKIWFPNRRMKWKK 163 3ProRQPKIWFPNRRMPWKK 164 Met-Arg RQIKIWFQNMRRKWKK 165 7Arg RQIRIWFQNRRMRWRR166 W/R RRWRRWWRRWWRRWRR 167

In another embodiment, a substrate composition of the invention includesa delivery agent which is a HIV trans-activator (TAT) protein or anactive fragment thereof. Such a delivery agent can include, for example,a sequence identical or similar to residues 47-57 or 47-59 of HIV TAT,see, e.g., Alan Frankel et al., Fusion Protein Comprising TAT-derivedTransport Moiert, U.S. Pat. No. 5,674,980 (Oct. 7, 1995); Alan Frankelet al., TAT-derived Transport Polypeptide Conjugates, U.S. Pat. No.5,747,641 (May 5, 1998); and Alan Frankel et al., TAT-derived TransportPolypeptides and Fusion Proteins, U.S. Pat. No. 5,804,604 (Sep. 8,1998). As an example, fusion proteins including residues 47-57 of HIVTAT (YGRKKRRQRRR; SEQ ID NO: 168) cross the plasma membrane of, forexample, human and murine cells in vitro and in vivo, see, e.g.,Schwartz and Zhang, supra, (2000); a variety of proteins from 15 to 120KDa have been shown to retain biological activity when fused to a HIVTAT delivery agent. An HIV TAT delivery agent can be positively chargedand can function, for example, in an energy-, receptor-, transporter-and endocytosis-independent manner to deliver a covalently linkedClostridial toxin substrate to, for example, 90-100% of target cells.Delivery agents using peptides derived from TAT and methods of usingsuch agents are described in, e.g., Frankel et al., supra, (1995);Frankel et al., supra, (1998); and Frankel et al., supra, (1998).

In another embodiment, a substrate composition of the invention also caninclude as a delivery agent a herpes simplex virus VP22 protein oractive fragment thereof. In an aspect of this embodiment, a substratecomposition of the invention includes an HSV type 1 (HSV-1) VP22 proteinor active fragment thereof. HSV VP22, a nuclear transcription factor,can cross the plasma membrane through non-classical endocytosis and canenter cells independent of GAP junctions and physical contacts. As afusion with a variety of different proteins, HSV VP22 results in uptakeinto cells of different types including terminally differentiated cellsand can function to deliver a linked Clostridial toxin substrate to, forexample, 90-100% of cultured cells. Delivery agents using peptidesderived from TAT and methods of using such agents are described in,e.g., Peter F. J. O'Hare & Gillian D. Elliott, Transport Proteins andTheir Uses, PCT Patent Publication No. WO97/05265 (Feb. 13, 1997); PeterF. J. O'Hare & Gillian D. Elliott, Fusion Proteins for Intracellular andIntercellular Transport and Their Uses, PCT Patent Publication No.WO98/32866 (Jul. 30, 1998); Peter F. J. O'Hare et al., Use of TransportProteins, U.S. Pat. No. 6,734,167 (May 11, 2004).

In another embodiment, a delivery agent useful in the inventioncorresponds to or is derived from a hydrophobic signal sequence. Such adelivery agent can be, for example, the Kaposi fibroblast growth factor(kFGF) or an active fragment thereof such as AAVALLPAVLLALLAP (SEQ IDNO: 169); human β3 integrin or an active fragment thereof; or anotherhydrophobic delivery agent such as one of those described in, e.g.,Dunican & Doherty, supra, (2001). Delivery agents using peptides derivedfrom hydrophobic signal sequences and methods of using such agents aredescribed in, e.g., Yao-Zhong Lin & Jack J. Hawiger, Method forimporting biologically active molecules into cells, U.S. Pat. No.5,807,746 (Sep. 15, 1998); Yao-Zhong Lin & Jack J. Hawiger, Method forimporting biologically active molecules into cells, U.S. Pat. No.6,043,339 (Mar. 28, 2000); Yao-Zhong Lin et al., Sequence and Method forGenetic Engineering of Proteins with Cell Membrane TranslocatingActivity, U.S. Pat. No. 6,248,558 (Jun. 19, 2001); Yao-Zhong Lin et al.,Sequence and Method for Genetic Engineering of Proteins with CellMembrane Translocating Activity, U.S. Pat. No. 6,432,680 (Aug. 13,2002); Jack J. Hawiger et al., Method for importing biologically activemolecules into cells, U.S. Pat. No. 6,495,518 (Dec. 17, 2002); andYao-Zhong Lin et al., Sequence and Method for Genetic Engineering ofProteins with Cell Membrane Translocating Activity, U.S. Pat. No.6,780,843 (Aug. 24, 2004).

In another embodiment, a delivery agent useful in the invention also canbe a synthetic sequence that shares one or more characteristics of anaturally occurring delivery agent such as, e.g., a protein transductiondomain (PTD). Such delivery agents include, but are not limited to, L-and D-arginine oligomers, for example, 9-mers of L- or D-arginine andrelated peptoids, see, e.g., Jonathan B. Rothbard & Paul A Wender,Method and Composition for Enhancing Transport Across BiologicalMembranes, U.S. Pat. No. 6,306,993 (Oct. 23, 2001); and Jonathan B.Rothbard & Paul A Wender, Method and Composition for Enhancing TransportAcross Biological Membranes, U.S. Pat. No. 6,495,663 (Dec. 17, 2002).Such delivery agents further include basic peptides and peptidomimetics;basic α-helical peptides and peptidomimetics; and peptides andpeptidomimetics with optimized arginine alignment or optimized α-helicalcharacter as compared to a naturally occurring protein transductiondomain such as residues 47-57 of HIV TAT, see, e.g., Rothbard & Wender,supra, (2001); and Rothbard & Wender, supra, (2002). The skilled personunderstands that these and other naturally occurring and syntheticdelivery agents can be useful in the substrate compositions of theinvention.

In another embodiment, a protein conjugate consisting of antibodydirected at a receptor on the plasma membrane and a Clostridial toxinsubstrate disclosed in the present specification can be introduced intoa cell. Delivery agents using antibodies and methods of using suchagents are described in, e.g., Pamela B. Davis et al., Fusion proteinsfor protein delivery, U.S. Pat. No. 6,287,817 (Sep. 11, 2001).

A delivery agent useful in the invention also can be an agent thatenables or enhances cellular uptake of a non-covalently associatedClostridial toxin substrate. In one embodiment, such a delivery agent ispeptide containing two independent domains: a hydrophobic domain and ahydrophilic domain.

In another embodiment, such a delivery agent is an MPG peptide, which isa peptide derived from both the nuclear localization sequence (NLS) ofSV40 large T antigen and the fusion peptide domain of HIV-1 gp41, see,e.g., Virginie Escriou et al., NLS bioconjugates for targetingtherapeutic genes to the nucleus, 55(2) Adv. Drug Deliv. Rev. 295-306(2003). In a further embodiment, such a delivery agent is an MPG peptidehaving the amino acid sequence GALFLGFLGAAGSTMGAWSQPKSKRKV (SEQ ID NO:172). In yet a further embodiment, such a delivery agent is anamphipathic peptide such as Pep-1. These and related delivery agentsthat function in the absence of covalent linkage and methods of usingsuch agents are described in, e.g., Gilles Divita et al,Peptide-mediated Transfection Agents and Methods of Use, U.S. Pat. No.6,841,535 (Jan. 11, 2005); Philip L Felgner and Olivier Zelphati,Intracellular Protein Delivery Compositions and Methods of Use, U.S.Patent Publication No. 2003/0008813); and Michael Karas IntracellularDelivery of Small Molecules, Proteins and Nucleic Acids, U.S. PatentPublication 2004/0209797 (Oct. 21, 2004). Such peptide delivery agentscan be prepared and used by standard methods and are commerciallyavailable, see, e.g. the Chariot™ Reagent (Active Motif, Carlsbad,Calif.); BioPORTER® Reagent (Gene Therapy Systems, Inc., San Diego,Calif.), BioTrek™ Protein Delivery Reagent (Stratagene, La Jolla,Calif.), and Pro-Ject™ Protein Transfection Reagent (PierceBiotechnology Inc., Rockford, Ill.).

Another aspect of the present invention provides expression constructthat allow for expression of a nucleic acid molecule encoding aClostridial toxin substrate disclosed in the present specification.These expression constructs comprise an open reading frame encoding aClostridial toxin substrate disclosed in the present specification,operably-linked to control sequences from an expression vector usefulfor expressing the Clostridial toxin substrate in a cell. The term“operably linked” as used herein, refers to any of a variety of cloningmethods that can ligate a nucleic acid molecule disclosed in the presentspecification into an expression vector such that a peptide encoded bythe composition is expressed when introduced into a cell.Well-established molecular biology techniques that may be necessary tomake an expression construct disclosed in the present specificationincluding, but not limited to, procedures involving polymerase chainreaction (PCR) amplification restriction enzyme reactions, agarose gelelectrophoresis, nucleic acid ligation, bacterial transformation,nucleic acid purification, nucleic acid sequencing are routineprocedures well within the scope of one skilled in the art and from theteaching herein. Non-limiting examples of specific protocols necessaryto make an expression construct are described in e.g., MOLECULAR CLONINGA LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004),which are hereby incorporated by reference. These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

A wide variety of expression vectors can be employed for expressing anopen reading frame encoding an Clostridial toxin substrate and includewithout limitation, viral expression vectors, prokaryotic expressionvectors and eukaryotic expression vectors including yeast, insect andmammalian expression vectors and generally are equivalent to theexpression vectors disclosed herein in Examples 4-6 and 8-14.Non-limiting examples of expression vectors, along with well-establishedreagents and conditions for making and using an expression constructfrom such expression vectors are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN,Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection,making and use of an appropriate expression vector are routineprocedures well within the scope of one skilled in the art and from theteachings herein.

It is envisioned that any of a variety of expression systems may beuseful for expressing construct compositions disclosed in the presentspecification. An expression system encompasses both cell-based systemsand cell-free expression systems. Cell-based systems include, withoutlimited, viral expression systems, prokaryotic expression systems, yeastexpression systems, baculoviral expression systems, insect expressionsystems and mammalian expression systems. Cell-free systems include,without limitation, wheat germ extracts, rabbit reticulocyte extractsand E. coli extracts. Expression using an expression system can includeany of a variety of characteristics including, without limitation,inducible expression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICALAPPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press,1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS.USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and MeenaRai & Harish Padh, Expression Systems for Production of HeterologousProteins, 80(9) CURRENT SCIENCE 1121-1128, (2001), which are herebyincorporated by reference. These protocols are routine procedures wellwithin the scope of one skilled in the art and from the teaching herein.

An expression construct comprising a nucleic acid molecule encoding aClostridial toxin substrate disclosed in the present specification canbe operationally-linked to a variety of regulatory elements that canpositively or negatively modulate, either directly or indirectly, theexpression of a nucleic acid molecule, such as, e.g., constitutive,tissue-specific, inducible or synthetic promoters and enhancers.Non-limiting examples of constitutive regulatory elements include, e.g.,the cytomegalovirus (CMV), herpes simplex virus thymidine kinase (HSVTK), simian virus 40 (SV40) early, 5′ long terminal repeat (LTR),elongation factor-1α (EF-1α) and polybiquitin (UbC) regulatory elements.Non-limiting examples of inducible regulatory elements useful in aspectsof the present invention include, e.g., chemical-inducible regulatoryelements such as, without limitation, alcohol-regulated,tetracycline-regulated, steroid-regulated, metal-regulated andpathogenesis-related; and physical-inducible regulatory elements suchas, without limitation, temperature-regulated and light-regulated. Suchinducible regulatory elements can be prepared and used by standardmethods and are commercially available, including, without limitation,tetracycline-inducible and tetracycline-repressible elements such as,e.g., Tet-On™ and Tet-Off™ (BD Biosciences-Clontech, Palo Alto, Calif.)and the T-REx™ (Tetracycline-Regulated Expression) and Flp-In™ T-REx™systems (Invitrogen, Inc., Carlsbad, Calif.); ecdysone-inducibleregulatory elements such as, e.g., the Complete Control® InducibleMammalian Expression System (Stratagene, Inc., La Jolla, Calif.);isopropyl β-D-galactopyranoside (IPTG)-inducible regulatory elementssuch as, e.g., the LacSwitch® ^(II) Inducible Mammalian ExpressionSystem (Stratagene, Inc., La Jolla, Calif.); and steroid-inducibleregulatory elements such as, e.g., the chimeric progesterone receptorinducible system, GeneSwitch™ (Invitrogen, Inc., Carlsbad, Calif.). Theskilled person understands that these and a variety of otherconstitutive and inducible regulatory systems are commercially availableor well known in the art and can be useful in the invention forcontrolling expression of a nucleic acid molecule which encodes aClostridial toxin substrate.

In an embodiment, a nucleic acid molecule encoding the Clostridial toxinsubstrate can optionally be linked to a regulatory element such as aconstitutive regulatory element.

In another embodiment, a nucleic acid molecule encoding the Clostridialtoxin substrate can optionally be linked to a regulatory element such asan inducible regulatory element. In an aspect of this embodiment,expression of the nucleic acid molecule is induced using, e.g.,tetracycline-inducible, ecdysone-inducible or steroid-inducible.

Aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that contain anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of said cells wherein said contacted cell population iscapable of Clostridial toxin intoxication, wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain and wherein greater than 50% of said cellpopulation comprises said cells containing said exogenous Clostridialtoxin substrate; exciting said fluorescent member; and determining thefluorescence of said contacted cell population relative to a controlcell population, where a difference in fluorescence of said contactedcell population as compared to said control cell population isindicative of Clostridial toxin activity.

Other aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that transientlycontain an exogenous Clostridial toxin substrate capable of beinglocalized to the plasma membrane of said cells wherein said contactedcell population is capable of Clostridial toxin intoxication, andwherein said substrate comprises a fluorescent member, a membranetargeting domain and a Clostridial toxin recognition sequence comprisinga cleavage site, where the cleavage site intervenes between saidfluorescent member and said membrane localization domain, whereingreater than 50% of said cell population comprises said cells containingsaid exogenous Clostridial toxin substrate; exciting said fluorescentmember; and determining the fluorescence of said contacted cellpopulation relative to a control cell population, where a difference influorescence of said contacted cell population as compared to saidcontrol cell population is indicative of Clostridial toxin activity.

Aspects of the present invention provide methods of determiningClostridial toxin activity by contacting with a sample a cellpopulation, the cell population comprising cells that stably contain anexogenous Clostridial toxin substrate capable of being localized to theplasma membrane of said cells wherein said contacted cell population iscapable of Clostridial toxin intoxication, and wherein said substratecomprises a fluorescent member, a membrane targeting domain and aClostridial toxin recognition sequence comprising a cleavage site, wherethe cleavage site intervenes between said fluorescent member and saidmembrane localization domain; exciting said fluorescent member; anddetermining the fluorescence of said contacted cell population relativeto a control cell population, where a difference in fluorescence of saidcontacted cell population as compared to said control cell population isindicative of Clostridial toxin activity.

In an embodiment, a method of determining Clostridial toxin activitycomprises the steps of contacting with a sample a cell population, thecell population comprising cells containing an exogenous Clostridialtoxin substrate capable of being localized to the plasma membrane ofsaid cells wherein said contacted cell population is capable ofClostridial toxin intoxication, and wherein said substrate comprises afluorescent member, a membrane targeting domain and a Clostridial toxinrecognition sequence comprising a cleavage site, where the cleavage siteintervenes between said fluorescent member and said membranelocalization domain; exciting said fluorescent member; and determiningthe fluorescence of said contacted cell population relative to a controlcell population, where a difference in fluorescence of said contactedcell population as compared to said control cell population isindicative of Clostridial toxin activity. In aspects of this embodiment,the Clostridial toxin substrate capable of being localized to the plasmamembrane can be, e.g., a BoNT/A substrate, a BoNT/B substrate, a BoNT/C1substrate, a BoNT/D substrate, a BoNT/E substrate, a BoNT/F substrate, aBoNT/G substrate or a TeNT substrate.

The methods disclosed in the present specification include, in part, aClostridial toxin substrate. In is envisioned that any and allClostridial toxin substrate disclosed in the present specification canbe used to practice the present methods. Thus, aspects of thisembodiment include nucleic acid molecules, such as, e.g., DNA and RNA,that encode a Clostridial toxin substrate disclosed in the presentspecification and peptide molecule or peptidomimetic comprising aClostridial toxin substrate disclosed in the present specification.Other aspects of this embodiment include, in part, a Clostridial toxinrecognition sequence including, without limitation, a BoNT/A toxinrecognition sequence, a BoNT/B toxin recognition sequence, a BoNT/C1toxin recognition sequence, a BoNT/D toxin recognition sequence, aBoNT/E toxin recognition sequence, a BoNT/F toxin recognition sequence,a BoNT/G toxin recognition sequence and a TeNT toxin recognitionsequence. Other aspects of this embodiment include, in part, a membranetargeting domain including, without limitation, naturally occurringmembrane targeting domains present in SNAP-25, naturally occurringSNAP-25 MTD variants, and non-naturally occurring SNAP-25 MTD variants,and SNAP-25 MTD peptidomimetics; and naturally occurring membranetargeting domains present in syntaxin, naturally occurring syntaxin MTDvariants, and non-naturally occurring syntaxin MTD variants and syntaxinMTD peptidomimetics. Other aspects of this embodiment include, in part,a fluorescent protein including, without limitation, wild typefluorescent proteins, naturally occurring variants, geneticallyengineered variants, active peptide fragments derived from Aequoreafluorescent proteins, Anemonia fluorescent proteins, Anthozoafluorescent proteins, Discosoma fluorescent proteins, Entacmeaefluorescent proteins, Heteractis fluorescent proteins, Montastreafluorescent proteins, Renilla fluorescent proteins, Zoanthus fluorescentproteins and fluorophore binding proteins. Non-limiting examples offluorescent proteins include, e.g., EBFP, ECFP, AmCyan, AcGFP, ZsGreen,Vitality® hrGFP, EGFP, Monster Green® hMGFP, EYFP, ZsYellow,DsRed-Express, DsRed2, DsRed, AsRed2 and HcRed1. Non-limiting examplesof fluorescent binding proteins include, e.g., a tetracysteine peptide,an AGT and a dehalogenase.

The methods disclosed in the present specification include, in part, acell capable of Clostridial toxin intoxication. In is envisioned thatany and all cells disclosed in the present specification can be used topractice the present methods. Thus, aspects of this embodiment includecells, such as, e.g., cells expressing one or more Clostridial toxinreceptors including, without limitation, a low affinity Clostridialtoxin receptor, a high affinity Clostridial toxin receptor, anendogenous Clostridial toxin receptor, an exogenous Clostridial toxinreceptors, a BoNT/A receptor, a BoNT/B receptor, a BoNT/C1 receptor, aBoNT/D receptor, a BoNT/E receptor, a BoNT/F receptor, a BoNT/G receptorand a TeNT receptor. Other aspects of this embodiment include cells,such as, e.g., neuronal cells including, without limitation, primaryneuronal cells; immortalized or established neuronal cells; transformedneuronal cells; neuronal tumor cells; stably and transiently transfectedneuronal cells expressing a Clostridial toxin receptor, and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman neuronal cells. Other aspects of this embodiment include cellsfrom, such as, e.g., neuronal cell lines including, without limitation,neuroblastoma cell lines, neuronal hybrid cell lines, spinal cord celllines, central nervous system cell lines, cerebral cortex cell lines,dorsal root ganglion cell lines, hippocampal cell lines andpheochromocytoma cell lines. Non-limiting examples of neuronal celllines include, e.g., neuroblastoma cell lines BE(2)-C, BE(2)-M17, C1300,CHP-212, CHP-126, IMR 32, KELLY, LA-N-2, MC-IXC, MHH-NB-11, N18Tg2,N1E-115, N4TG3, Neuro-2A, NB41A3, NS20Y, SH-SY5Y, SIMA, SK-N-DZ,SK-N-F1, SK-N-MC and SK-N-SH; neuroblastoma/glioma hybrid cell linesN18, NG108-15 and NG115-401L; neuroblastoma/motor neuron hybrid celllines NSC-19 and NSC-32; neuroblastoma/root ganglion neuron hybrid celllines F11, ND-C, ND-E, ND-U1, ND3, ND7/23, ND8/34, ND8/47, ND15 andND27; the neuroblastoma/hippocampal neuron hybrid cell line HN-33;spinal cord cell lines TE 189.T and M4b; cerebral cortex cell lines CNh,HCN-1a and HCN-2; dorsal root ganglia cell line G4b; hippocampal celllines HT-4, HT-22 and HN33; FGFR3 expressing cell lines H929, JIM-3,KMS-11, KMS-18, LB278, LB375, LB1017, LB2100, LP-1, OPM-2, PCL1 andUTMC-2. In further aspects of this embodiment, an FGFR3 expressing cellcan be, e.g., H929, JIM-3, KMS-11, KMS-18, LB278, LB375, LB1017, LB2100,LP-1, OPM-2, PCL1 UTMC-2, B9, TC, L6 and CFK2. Other aspects of thisembodiment include cells, such as, e.g., non-neuronal cells including,without limitation, primary non-neuronal cells; immortalized orestablished non-neuronal cells; transformed non-neuronal cells;non-neuronal tumor cells; stably and transiently transfectednon-neuronal cells expressing a Clostridial toxin receptor, and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman non-neuronal cells. Other aspects of this embodiment includecells, such as, e.g., non-neuronal cells useful in aspects of theinvention further include, without limitation, anterior pituitary cells;adrenal cells, pancreatic cells, ovarian cells, kidney cells, such as,e.g., HEK293, stomach cell, blood cells, epithelial cells, fibroblasts,thyroid cells, chondrocytes, muscle cells, hepatocytes, glandular cellsand cells involved in glucose transporter (GLUT4) translocation.

The methods disclosed in the present specification include, in part, asample. As used herein, the term “sample” means any biological matterthat contains or potentially contains an active Clostridial toxin. Avariety of samples can be assayed according to a method disclosed in thepresent specification including, without limitation, purified, partiallypurified, or unpurified Clostridial toxin; recombinant single chain ordi-chain toxin with a naturally or non-naturally occurring sequence;recombinant Clostridial toxin with a modified protease specificity;recombinant Clostridial toxin with an altered cell specificity; chimerictoxin containing structural elements from multiple Clostridial toxinspecies or subtypes; bulk Clostridial toxin; formulated Clostridialtoxin product, including, e.g., formulated BoNT/A and BoNT/E products;and foods; cells or crude, fractionated or partially purified celllysates, for example, engineered to include a recombinant nucleic acidencoding a Clostridial toxin; bacterial, baculoviral and yeast lysates;raw, cooked, partially cooked or processed foods; beverages; animalfeed; soil samples; water samples; pond sediments; lotions; cosmetics;and clinical formulations. It is understood that the term sampleencompasses tissue samples, including, without limitation, mammaliantissue samples, livestock tissue samples such as sheep, cow and pigtissue samples; primate tissue samples; and human tissue samples. Suchsamples encompass, without limitation, intestinal samples such as infantintestinal samples, and tissue samples obtained from a wound. Asnon-limiting examples, a method of the invention can be useful fordetermining the presence or activity of a Clostridial toxin in a food orbeverage sample; to assay a sample from a human or animal, for example,exposed to a Clostridial toxin or having one or more symptoms of aClostridial toxin; to follow activity during production and purificationof Clostridial toxin; or to assay formulated Clostridial toxin productssuch as pharmaceuticals or cosmetics.

The methods disclosed in the present specification include, in part,determining the Clostridial toxin activity from a sample by detectingthe fluorescence intensity of a Clostridial toxin substrate from a cellcontacted population with the sample. As used herein, with regard todetecting fluorescence intensity the term “contacted cell population”means both detecting the fluorescence intensity of the contacted cellpopulation in the aggregate or detecting the fluorescence intensity ofindividual cells comprising the contacted cell population and analysisthe obtained data individually or in the aggregate. A variety of meanscan be useful for determining the fluorescence intensity of aClostridial toxin substrate contained in a cell contacted populationwith sample, including, without limitation, detecting the localizationof fluorescence intensity within the contacted cell population,detecting fluorescence intensity in the contacted cell population overtime, detecting fluorescence intensity in the contacted cell populationover time and comparing fluorescence intensity detected in the contactedcell population relative to the fluorescence intensity detected in acontrol cell population. As used herein, the term “control cellpopulation” means a cell population of the same or similar type as thecontacted cell population and grown under the same conditions but whichis not contacted with any sample or is contacted with a defined negativesample or a defined positive sample. As used herein, with regard todetecting fluorescence intensity the term “control cell population”means both detecting the fluorescence intensity of the control cellpopulation in the aggregate or detecting the fluorescence intensity ofindividual cells comprising the control cell population and analysis theobtained data individually or in the aggregate. One skilled in the artunderstands that a variety of control cell populations are useful in themethods of the invention and that a control cell population can be apositive control cell population or a negative control cell population.A control cell population can be, for example, a negative control cellpopulation such as a similar or identical cell population comprisingcells containing the same or similar Clostridial toxin substrate that iscontacted with a similar, defined negative sample, which is known tolack active Clostridial toxin, or that is not contacted with any sample.A control cell population also can be, for example, a positive controlcell population such as a cell population comprising cells containingone or both cleavage products that result from proteolysis of theClostridial toxin substrate at the cleavage site or a cell populationcomprising cells containing the same or similar substrate contacted witha defined positive sample, which is known to include active Clostridialtoxin.

Fluorescence intensity and, hence, Clostridial toxin activity, can bedetected by a variety of means, for example, by detecting increasedfluorescence intensity of the cytoplasmic substrate from the contactedcell population; decreased fluorescence intensity of themembrane-localized substrate from the contacted cell population; a shiftin fluorescence intensity of the membrane-localized substrate to thecytoplasmic substrate; a decreased fluorescence intensity found in acertain sub-cellular area of cells comprising the contacted cellpopulation; an increased fluorescence intensity found in a certainsub-cellular area of cells comprising the contacted cell population. Inaspects of this embodiment, a decreased fluorescence intensity can be,e.g., at least two-fold, at least three-fold, at least four-fold, atleast five-fold, at least ten-fold, at least twenty-fold or morerelative to fluorescence intensity at the same wavelength of the samecell population detected at a different time point, or relative tofluorescence intensity at the same wavelength of a similar cellpopulation not contacted with a sample, such as, e.g., a control cellpopulation. In other aspects of this embodiment, a decreasedfluorescence intensity can be, e.g., at most two-fold, at mostthree-fold, at most four-fold, at most five-fold, at most ten-fold, atmost twenty-fold relative to fluorescence intensity at the samewavelength of the same cell population detected at a different timepoint, or relative to fluorescence intensity at the same wavelength of asimilar cell population not contacted with a sample, such as, e.g., acontrol cell population. In yet other aspects of this embodiment, anincreased fluorescence intensity can be, e.g., at least two-fold, atleast three-fold, at least four-fold, at least five-fold, at leastten-fold, at least twenty-fold or more relative to fluorescenceintensity at the same wavelength of the same cell population detected ata different time point, or relative to fluorescence intensity at thesame wavelength of a similar cell population not contacted with asample, such as, e.g., a control cell. In yet other aspects of thisembodiment, an increased fluorescence intensity can be, e.g., at mosttwo-fold, at most three-fold, at most four-fold, at most five-fold, atmost ten-fold, at most twenty-fold relative to fluorescence intensity atthe same wavelength of the same cell population detected at a differenttime point, or relative to fluorescence intensity at the same wavelengthof a similar cell population not contacted with a sample, such as, e.g.,a control cell.

It is understood that the relevant fluorescence intensities are detectedat the appropriate wavelength or range of wavelengths. As an example,where the fluorescence intensity of the membrane-localized substrate isdetected, the appropriate wavelength is at or near the emission maximaof the fluorescent member comprising the membrane-localized substrate,or is a range of wavelengths encompassing or near to the emission maximaof the fluorescent member comprising the membrane-localized substrate.It is recognized that changes in the absolute amount of Clostridialtoxin substrate in the cell, fluorescence intensity, and turbidity orother background absorbance at the excitation wavelength effects thefluorescence intensities of the fluorescent member comprising theClostridial toxin substrate roughly in parallel. Thus, it is understoodthat a ratio of emission fluorescence intensities is independent of theabsolute amount of substrate, fluorescence intensity, and turbidity orother background absorbance, and can be a useful indicator ofClostridial toxin activity.

In one embodiment, Clostridial toxin activity from a sample isdetermined by detecting the fluorescence intensity of themembrane-localized Clostridial toxin substrate from the contacted cellpopulation, where a decreased in fluorescence intensity over time of themembrane-localized substrate from the contacted cell population isindicative of Clostridial toxin activity. Detection of fluorescenceintensity can be practiced as “fixed-time” assays or as continuous-timeassays and comparisons can be made using different time points takenfrom the same contacted cell population or relative to a control cellpopulation. Thus, aspect of this embodiment include detecting thefluorescence intensity of the membrane-localized Clostridial toxinsubstrate in, e.g., at least two different time points, at least threedifferent time points, at least four different time points, at leastfive different time points, at least ten different time points and atleast 20 different time points. Other aspects of this embodiment includedetecting the fluorescence intensity of the membrane-localizedClostridial toxin substrate over time intervals that are, e.g., no morethan 1 minute apart, no more than 5 minutes apart, no more than 10minutes apart, no more than 15 minutes apart, no more than 30 minutesapart and no more than 30 minutes apart. Other aspects of thisembodiment include detecting the fluorescence intensity of themembrane-localized Clostridial toxin substrate over time intervals thatare, e.g., no less than 15 minutes apart, no less than 30 minutes apart,no less than 45 minutes apart, no less than 60 minutes apart, no lessthan 90 minutes apart and no less than 120 minutes apart. Still otheraspects of this embodiment include detecting the fluorescence intensityof the membrane-localized Clostridial toxin substrate continuously overtime for, e.g., at most about 5 minutes, at most about 10 minutes, atmost about 15 minutes, at most about 30 minutes, at most about 45minutes, at most about 60 minutes, at most about 90 minutes and at mostabout 120 minutes. Still other aspects of this embodiment includedetecting the fluorescence intensity of the membrane-localizedClostridial toxin substrate continuously over time for, e.g., at leastabout 15 minutes, at least about 30 minutes, at least about 45 minutes,at least about 60 minutes, at least about 90 minutes and at least about120 minutes.

In another embodiment, Clostridial toxin activity from a sample isdetermined by detecting the fluorescence intensity of the cytoplasmicClostridial toxin substrate from the contacted cell population, whereincreased fluorescence intensity over time of the cytoplasmic substratefrom the contacted cell population is indicative of Clostridial toxinactivity. Detection of fluorescence intensity can be practiced as a“fixed-time” assay or as a continuous-time assay and comparisons can bemade using different time points taken from the same contacted cellpopulation or relative to a control cell population . . . . Thus, aspectof this embodiment include detecting the fluorescence intensity of thecytoplasmic Clostridial toxin substrate in, e.g., at least two differenttime points, at least three different time points, at least fourdifferent time points, at least five different time points, at least tendifferent time points and at least 20 different time points. Otheraspects of this embodiment include detecting the fluorescence intensityof the cytoplasmic Clostridial toxin substrate over time intervals thatare, e.g., no more than 1 minute apart, no more than 5 minutes apart, nomore than 10 minutes apart, no more than 15 minutes apart, no more than30 minutes apart and no more than 30 minutes apart. Other aspects ofthis embodiment include detecting the fluorescence intensity of thecytoplasmic Clostridial toxin substrate over time intervals that are,e.g., no less than 15 minutes apart, no less than 30 minutes apart, noless than 45 minutes apart, no less than 60 minutes apart, no less than90 minutes apart and no less than 120 minutes apart. Still other aspectsof this embodiment include detecting the fluorescence intensity of thecytoplasmic Clostridial toxin substrate continuously over time for,e.g., at most about 5 minutes, at most about 10 minutes, at most about15 minutes, at most about 30 minutes, at most about 45 minutes, at mostabout 60 minutes, at most about 90 minutes and at most about 120minutes. Still other aspects of this embodiment include detecting thefluorescence intensity of the cytoplasmic Clostridial toxin substratecontinuously over time for, e.g., at least about 15 minutes, at leastabout 30 minutes, at least about 45 minutes, at least about 60 minutes,at least about 90 minutes and at least about 120 minutes.

In another embodiment, Clostridial toxin activity from a sample isdetermined by detecting the fluorescence intensity of themembrane-localized Clostridial toxin substrate from the contacted cellpopulation, where a decreased in fluorescence intensity of themembrane-localized substrate from the contacted cell population, ascompared to the control cell population, is indicative of Clostridialtoxin activity. It is understood that fluorescence intensity can bedetected from a single time point or a plurality of time points. It isenvisioned that comparison of the fluorescence intensity detected fromthe contacted cell population to the fluorescence intensity detectedfrom the control cell population can be made using the values obtainedfrom the same, or similar time point or from different time points.Thus, aspect of this embodiment include detecting the fluorescenceintensity of the membrane-localized Clostridial toxin substrate from thecontacted cell population and control cell population in, e.g., at leastone different time point, at least two different time points, at leastthree different time points, at least four different time points, atleast five different time points, at least ten different time points andat least 20 different time points. Other aspects of this embodiment caninclude comparison of the fluorescence intensity detected from thecontacted cell population obtained from a single time point to thefluorescence intensity detected from the control cell populationobtained, e.g., at the same time point, at a similar time point, at atime point later than the time point obtained from the contact cellpopulation, at a time point earlier than the time point obtained fromthe contact cell population, at a plurality time points later than thetime point obtained from the contact cell population, at a pluralitytime points earlier than the time point obtained from the contact cellpopulation and at a plurality time point both later than and earlierthan the time point obtained from the contact cell population, Otheraspects of this embodiment can include comparison of the fluorescenceintensity detected from the contacted cell population obtained from aplurality of time points to the fluorescence intensity detected from thecontrol cell population obtained, e.g., from a single time point, at thesame time points, at a similar time points, at a time point later thanthe time points obtained from the contact cell population, at a timepoint earlier than the time points obtained from the contact cellpopulation, at a plurality time points later than the time pointsobtained from the contact cell population, at a plurality time pointsearlier than the time points obtained from the contact cell populationand at a plurality time point both later than and earlier than the timepoints obtained from the contact cell population,

In another embodiment, Clostridial toxin activity from a sample isdetermined by detecting the fluorescence intensity of the cytoplasmicClostridial toxin substrate from the contacted cell population, where anincreased in fluorescence intensity of the cytoplasmic substrate fromthe contacted cell population, as compared to the control cellpopulation, is indicative of Clostridial toxin activity. It isunderstood that fluorescence intensity can be detected from a singletime point or a plurality of time points. It is envisioned thatcomparison of the fluorescence intensity detected from the contactedcell population to the fluorescence intensity detected from the controlcell population can be made using the values obtained from the same, orsimilar time point or from different time points. Thus, aspect of thisembodiment include detecting the fluorescence intensity of thecytoplasmic Clostridial toxin substrate from the contacted cellpopulation and control cell population in, e.g., at least one differenttime point, at least two different time points, at least three differenttime points, at least four different time points, at least fivedifferent time points, at least ten different time points and at least20 different time points. Other aspects of this embodiment can includecomparison of the fluorescence intensity detected from the contactedcell population obtained from a single time point to the fluorescenceintensity detected from the control cell population obtained, e.g., atthe same time point, at a similar time point, at a time point later thanthe time point obtained from the contact cell, population at a timepoint earlier than the time point obtained from the contact cellpopulation, at a plurality time points later than the time pointobtained from the contact cell population, at a plurality time pointsearlier than the time point obtained from the contact cell populationand at a plurality time point both later than and earlier than the timepoint obtained from the contact cell population, Other aspects of thisembodiment can include comparison of the fluorescence intensity detectedfrom the contacted cell population obtained from a plurality of timepoints to the fluorescence intensity detected from the control cellpopulation obtained, e.g., from a single time point, at the same timepoints, at a similar time points, at a time point later than the timepoints obtained from the contact cell population, at a time pointearlier than the time points obtained from the contact cell population,at a plurality time points later than the time points obtained from thecontact cell population, at a plurality time points earlier than thetime points obtained from the contact cell population and at a pluralitytime point both later than and earlier than the time points obtainedfrom the contact cell population,

In another embodiment, Clostridial toxin activity from a sample isdetermined by detecting the fluorescence intensity of the Clostridialtoxin substrate found in a certain sub-cellular area of cells comprisingthe contacted cell population. Aspects of this embodiment can includedetecting a decreased in fluorescence intensity of the Clostridial toxinsubstrate found in a certain sub-cellular area of the cells comprisingthe contacted cell population, detecting an increase in fluorescenceintensity of the Clostridial toxin substrate found in a certainsub-cellular area of the cells comprising the contacted cell population,or both. In other aspects of this embodiment, the sub-cellular regioncan be, e.g., the cell membrane, the cytoplasm, the endoplasmicreticulum, an organelle and a vesicle.

In a method of the invention, fluorescence of a contacted cell typicallyis determined using a fluorimeter. In general, excitation radiation froman excitation source having a first wavelength passes through excitationoptics. The excitation optics cause the excitation radiation to excitethe substrate in the cell. In response, the fluorescent member in thesubstrate emit radiation which has a wavelength that is different fromthe excitation wavelength. Collection optics then collect the emission;if desired, the device includes a temperature controller to maintain thecell at a specific temperature while being scanned. If desired, a multiaxis translation stage moves a microtiter plate containing a pluralityof samples in order to position different wells to be exposed. It isunderstood that the multi-axis translation stage, temperaturecontroller, auto-focusing feature, and electronics associated withimaging and data collection can be managed by the appropriate digitalcomputer.

It is further understood that the methods of the invention can beautomated and can be configured in a high throughput or ultrahigh-throughput format using, without limitation, 96-well, 384-well or1536-well plates. As one non-limiting example, fluorescence emission canbe detected using the SpectraMax M5 microplate reader (MolecularDevices, Sunnyvale, Calif.), a dual-monochromator, multi-detectionmicroplate reader with a wavelength range of 250-850 nm and a 6-384microplate reading capability. As another non-limiting example,fluorescence emission can be detected using the Typhoon™ 9410 system(Amersham Biosciences, Piscataway, N.J.). Designed for microplateassays, this system utilizes is capable of excitation fluorescence at488 nm, 532 nm or 633 nm and has a semiconfocal optimal system with acharge coupled device (CCD) camera to illuminate and image the entireplate. The FPM-2 96-well plate reader (Folley Consulting and Research,Round Lake, Ill.) also can be useful in detecting fluorescence emissionin the methods of the invention. One skilled in the art understands thatthese and other automated systems with the appropriate spectroscopiccompatibility such as the ECLIPSE cuvette reader (Varian-Cary; WalnutCreek, Calif.) and the FLIPR® and Gemini XPS spectrofluorimeter systems(Molecular Devices, Sunnyvale, Calif.).

It is envisioned that a variety of conditions suitable for determiningClostridial toxin activity in a sample can be useful according to themethods disclosed in the present specification. In aspects of thisembodiment, conditions suitable for determining Clostridial toxinactivity can be provided such that, e.g., at least 10% of the substrateis cleaved, at least 20% of the substrate is cleaved, at least 30% ofthe substrate is cleaved, at least 40% of the substrate is cleaved, atleast 50% of the substrate is cleaved, at least 60% of the substrate iscleaved, at least 70% of the substrate is cleaved, at least 80% of thesubstrate is cleaved or at least 90% of the substrate is cleaved. Inother aspects of this embodiment, conditions suitable for determiningClostridial toxin activity can be provided such that, e.g., at most 10%of the substrate is cleaved, at most 20% of the substrate is cleaved, atmost 30% of the substrate is cleaved, at most 40% of the substrate iscleaved, at most 50% of the substrate is cleaved, at most 60% of thesubstrate is cleaved, at most 70% of the substrate is cleaved, at most80% of the substrate is cleaved or at most 90% of the substrate iscleaved. In another aspect of this embodiment, conditions suitable fordetermining Clostridial toxin activity can be provided such that 100% ofthe substrate is cleaved. In another aspect of this embodiment, theconditions suitable for determining Clostridial toxin activity areprovided such that the assay is linear. In another aspect of thisembodiment, the conditions suitable for determining Clostridial toxinactivity are provided such that the assay is non-linear.

Clostridial toxins are zinc metalloproteases, and a source of zinc, suchas zinc chloride or zinc acetate, typically in the range of 1 to 500 μM,for example, 5 to 10 μM can be included, if desired, as part of theconditions suitable for determining Clostridial toxin activity. Oneskilled in the art understands that zinc chelators such as EDTAgenerally are excluded from a buffer for determining the presence oractivity of a Clostridial toxin.

The concentration of purified or partially purified Clostridial toxin tobe assayed in a method of the invention generally is in the range ofabout 0.0001 ng/ml to 500 μg/ml toxin, for example, about 0.0001 ng/mlto 50 μg/ml toxin, 0.001 ng/ml to 500 μg/ml toxin, 0.001 ng/ml to 50μg/ml toxin, 0.0001 to 5000 ng/ml toxin, 0.001 ng/ml to 5000 ng/ml, 0.01ng/ml to 5000 ng/ml, 0.1 ng/ml to 5000 ng/ml, 0.1 ng/ml to 500 ng/ml,0.1 ng/ml to 50 ng/ml, 1 ng/ml to 5000 ng/ml, 1 ng/ml to 500 ng/ml, 1ng/ml to 50 ng/ml, 10 ng/ml to 5000 ng/ml, 10 ng/ml to 500 ng/ml, 50ng/ml to 5000 ng/ml, 50 ng/ml to 500 ng/ml or 100 ng/ml to 5000 ng/mltoxin, which can be, for example, purified recombinant dichain or singlechain toxin or formulated Clostridial toxin product containing humanserum albumin and excipients. In aspects of this embodiment, theconcentration of purified or partially purified Clostridial toxinassayed results in cleavage of, e.g., at least 10% of the totalsubstrate present, at least 20% of the total substrate present, at least30% of the total substrate present, at least 40% of the total substratepresent, at least 50% of the total substrate present, at least 60% ofthe total substrate present, at least 70% of the total substratepresent, at least 80% of the total substrate present or at least 90% ofthe total substrate present. In further aspects of this embodiment, theconcentration of purified or partially purified Clostridial toxinassayed results in cleavage of, e.g., at most 10% of the total substratepresent, at most 20% of the total substrate present, at most 30% of thetotal substrate present, at most 40% of the total substrate present, atmost 50% of the total substrate present, at most 60% of the totalsubstrate present, at most 70% of the total substrate present, at most80% of the total substrate present or at most 90% of the total substratepresent. In another aspect of this embodiment, the concentration ofpurified or partially purified Clostridial toxin assayed results incleavage of 100% of the total substrate present.

The concentration of purified or partially purified Clostridial toxinassayed in a method of the invention can be, for example, in the rangeof about 0.1 pM to 500 μM, 0.1 pM to 100 μM, 0.1 pM to 10 μM, 0.1 pM to1 μM, 0.1 pM to 500 nM, 0.1 pM to 100 nM, 0.1 pM to 10 nM, 0.1 pM to 1nM, 0.1 pM to 500 pM, 0.1 pM to 100 pM, 0.1 pM to 50 pM, 0.1 pM to 10pM, 1 pM to 500 μM, 1 pM to 100 μM, 1 pM to 10 μM, 1 pM to 1 μM, 1 pM to500 nM, 1 pM to 100 nM, 1 pM to 10 nM, 1 pM to 1 nM, 1 pM to 500 pM, 1pM to 100 pM, 1 pM to 50 pM, 1 pM to 10 pM, 10 pM to 500 μM, 10 pM to100 μM, 10 pM to 10 μM, 10 pM to 10 μM, 10 pM to 500 nM, 10 pM to 100nM, 10 pM to 10 nM, 10 pM to 1 nM, 10 pM to 500 pM, 10 pM to 100 pM, 10pM to 50 pM, 100 pM to 500 μM, 100 pM to 100 μM, 100 pM to 10 μM, 100 pMto 1 μM, 100 pM to 500 nM, 100 pM to 100 nM, 100 pM to 10 nM, 100 pM to1 nM, 100 pM to 500 pM 1 nM to 500 μM, 1 nM to 100 μM, 1 nM to 10 μM, 1nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10nM, 3 nM to 100 nM toxin, which can be, for example, purified native orrecombinant light chain or dichain toxin or formulated Clostridial toxinproduct containing human serum albumin and excipients. One skilled inthe art understands that the concentration of purified or partiallypurified Clostridial toxin will depend on the serotype of the toxinassayed, as well as the purity or recombinant sequence of the toxin, thepresence of inhibitory components, and the assay conditions. It isadditionally understood that purified, partially purified or crudesamples can be diluted to within a convenient range for assaying forClostridial toxin activity against a standard curve. Similarly, it isunderstood that a sample can be diluted, if desired, such that the assayis linear. In aspects of this embodiment, the concentration of purifiedor partially purified Clostridial toxin assayed results in cleavage of,e.g., at least 10% of the total substrate present, at least 20% of thetotal substrate present, at least 30% of the total substrate present, atleast 40% of the total substrate present, at least 50% of the totalsubstrate present, at least 60% of the total substrate present, at least70% of the total substrate present, at least 80% of the total substratepresent at least 90% of the total substrate present. In further aspectsof this embodiment, the concentration of purified or partially purifiedClostridial toxin assayed results in cleavage of, e.g., at most 10% ofthe total substrate present, at most 20% of the total substrate present,at most 30% of the total substrate present, at most 40% of the totalsubstrate present, at most 50% of the total substrate present, at most60% of the total substrate present, at most 70% of the total substratepresent, at most 80% of the total substrate present at most 90% of thetotal substrate present. In another aspect of this embodiment, theconcentration of purified or partially purified Clostridial toxinassayed results in cleavage of 100% of the total substrate present.

In still another embodiment, it is envisioned that any and alltemperatures that allow the function of a Clostridial activity assay canbe used in methods disclosed in the present specification. Assaytemperatures can be varied as appropriate by one skilled in the art andgenerally depend, in part, on the concentration, purity and activity ofthe Clostridial toxin, the sample to be assayed, the assay time or theconvience of the artisan. Thus, an assay temperature should not be aslow as to cause the solution to freeze and should not be as high as todenature the Clostridial toxin, the Clostridial toxin substratedisclosed in the present specification. In an aspect of this embodiment,the assay is performed within a temperature range above 0° C., but below40° C. In another aspect of this embodiment, the assay is performedwithin a temperature range of about 4° C. to about 37° C. In yet anotheraspect of this embodiment, the assay is performed within a temperaturerange of about 2° C. to 10° C. In yet another aspect of this embodiment,the assay is performed at about 4° C. In still another aspect of thisembodiment, the assay is performed within a temperature range of about10° C. to about 18° C. In still another aspect of this embodiment, theassay is performed at about 16° C. In yet another aspect of thisembodiment, the assay is performed within a temperature range of about18° C. to about 32° C. In yet another aspect of this embodiment, theassay is performed at about 20° C. In another aspect of this embodiment,the assay is performed within a temperature range of about 32° C. toabout 40° C. In another aspect of this embodiment, the assay isperformed at about 37° C. In aspects of this embodiment, the amount ofClostridial toxin substrate cleaved within a temperature range is, e.g.,at least 10% of the total substrate present, at least 20% of the totalsubstrate present, at least 30% of the total substrate present, at least40% of the total substrate present, at least 50% of the total substratepresent, at least 60% of the total substrate present, at least 70% ofthe total substrate present, at least 80% of the total substrate presentor at least 90% of the total substrate present. In further aspects ofthis embodiment, the amount of Clostridial toxin substrate cleavedwithin a temperature range is, e.g., at most 10% of the total substratepresent, at most 20% of the total substrate present, at most 30% of thetotal substrate present, at most 40% of the total substrate present, atmost 50% of the total substrate present, at most 60% of the totalsubstrate present, at most 70% of the total substrate present, at most80% of the total substrate present or at most 90% of the total substratepresent. In another aspect of this embodiment, the amount of Clostridialtoxin substrate cleaved within a temperature range is 100%.

In still another embodiment, it is foreseen that any and all timessufficient for the detection of the presence of Clostridial toxinsubstrate cleavage products can be used in methods disclosed in thepresent specification. Assay times can be varied as appropriate by theskilled artisan and generally depend, in part, on the concentration,purity and activity of the Clostridial toxin, the sample to be assayed,incubation temperature or the convience of the artisan. Assay timesgenerally vary, without limitation, in the range of about 15 minutes toabout 4 hours, 30 minutes to 8 hours, 1 hours to 12 overs, 2 hours to 24hours, 4 hours to 48 hours, 6 hours to 72 hours. In aspects of thisembodiment, the amount of Clostridial toxin substrate cleaved during anassay time is, e.g., at least 10% of the total substrate present, atleast 20% of the total substrate present, at least 30% of the totalsubstrate present, at least 40% of the total substrate present, at least50% of the total substrate present, at least 60% of the total substratepresent, at least 70% of the total substrate present, at least 80% ofthe total substrate present or at least 90% of the total substratepresent. In further aspects of this embodiment, the amount ofClostridial toxin substrate cleaved during an assay time is, e.g., atmost 10% of the total substrate present, at most 20% of the totalsubstrate present, at most 30% of the total substrate present, at most40% of the total substrate present, at most 50% of the total substratepresent, at most 60% of the total substrate present, at most 70% of thetotal substrate present, at most 80% of the total substrate present orat most 90% of the total substrate present. In another aspect of thisembodiment, the amount of Clostridial toxin substrate cleaved during anassay time is 100%. It is understood that assays can be terminated, ifdesired, prior to exciting the fluorescent member.

Aspects of the present invention can also be describes as follows:

-   1. A composition comprising a cell population, said population    comprising cells containing an exogenous Clostridial toxin substrate    capable of being localized to the plasma membrane of said cells;    wherein said cells are capable of Clostridial toxin intoxication;    wherein said exogenous Clostridial toxin substrate comprises a    fluorescent member, a membrane targeting domain and a Clostridial    toxin recognition sequence comprising a cleavage site, where the    cleavage site intervenes between said fluorescent member and said    membrane localization domain; and wherein greater than 50% of said    cell population comprises said cells containing said exogenous    Clostridial toxin substrate.-   2. The composition according to 1, wherein said cells transiently    contains said exogenous Clostridial toxin substrate.-   3. The composition according to 1, wherein said cells stably    contains said exogenous Clostridial toxin substrate.-   4. The composition according to 1, wherein said substrate is    expressed from a nucleic acid molecule.-   5. The composition according to 4, wherein said nucleic acid    molecule comprises a viral expression construct encoding a    Clostridial toxin substrate.-   6. The composition according to 1, wherein said toxin substrate is    introduced into said cell using a protein delivery method.-   7. The composition according to 1, wherein said cell is a neuronal    cell.-   8. The composition according to 7, wherein said neuronal cell is    selected from the group consisting of a primary neuronal cell, an    immortalized neuronal cell and a transformed neuronal cell.-   9. The composition according to 7, wherein said neuronal cell is    selected from the group consisting of a neuroblastoma cell, a    neuronal hybrid cell, a spinal cord cell, a central nervous system    cell, a cerebral cortex cell, a dorsal root ganglion cell, a    hippocampal cell and a pheochromocytoma cell.-   10. The composition according to 7, wherein said neuronal cell is    selected from the group consisting of Neuro-2a, SH-SY5Y, NG108-C15,    N1E-115, ND8/34 and SK-N-DZ.-   11. The composition according to 1, wherein said cell is a    non-neuronal cell.-   12. The composition according to 11, wherein said non-neuronal cell    is selected from the group consisting of a primary non-neuronal    cell, an immortalized non-neuronal cell and a transformed    non-neuronal cell.-   13. The composition according to 11, wherein said non-neuronal cell    is selected from the group consisting of an anterior pituitary cell,    an adrenal cell, a pancreatic cell, an ovarian cell, a kidney cell,    a stomach cell, a blood cell, an epithelial cell, a fibroblast, a    thyroid cell, a chondrocyte, a muscle cell, a hepatocyte, a    glandular cell.-   14. The composition according to 11, wherein said kidney cell is    HEK-293.-   15. The composition according to 1, wherein said cell is sensitive    to said intoxication at Clostridial toxin concentrations of 2 nM and    below.-   16. The composition according to 1, wherein said cell is sensitive    to said intoxication at Clostridial toxin concentrations of 0.1 nM    and below.-   17. The composition according to 1, wherein said cell is sensitive    to said intoxication at Clostridial toxin concentrations of 0.02 nM    and below.-   18. The composition according to 1, wherein said fluorescent member    is a fluorescent protein.-   19. The composition according to 18, wherein said fluorescent    protein is selected from the group consisting of a green fluorescent    protein, a blue fluorescent protein, a cyan fluorescent protein, a    yellow fluorescent protein and a red fluorescent protein.-   20. The composition according to 1, wherein said fluorescent protein    is a fluorophore binding protein.-   21. The composition according to 20, wherein said fluorophore    binding protein is selected from the group consisting of a    tetracysteine peptide, an AGT and a dehalogenase.-   22. The composition according to 21, wherein said tetracysteine    peptide binds to a fluorophore selected from the group consisting of    a nonfluorescent biarsenical derivitive of fluorescein and a    nonfluorescent biarsenical derivitive of resorufin.-   23. The composition according to 21, wherein said AGT binds to a    fluorophore selected from the group consisting of a para-benzyl    guanine diethylaminocoumarin, a para-benzyl guanine    diacetylfluorescein, a para-benzyl guanine dyomic DY-505-05, a    para-benzyl guanine ATTO 488, a para-benzyl guanine ATTO 532, a    para-benzyl guanine dyomic DY-547, a para-benzyl guanine    tetramethylrhodamine, a para-benzyl guanine ATTO 600, a para-benzyl    guanine dyomic DY-632, a para-benzyl guanine dyomic DY-647, a    para-benzyl guanine dyomic DY-732 and a para-benzyl guanine dyomic    DY-747.-   24. The composition according to 21, wherein said dehalogenase binds    to a fluorophore selected from the group consisting of a HaloTag    Coumarian, a HaloTag diAcFAM and a HaloTag TMR.-   25. The composition according to 1, wherein said membrane targeting    domain comprises a interhelical loop region of SNAP-25.-   26. The composition according to 25, wherein said interhelical loop    region of SNAP-25 comprises at least 5 residues from amino acids    85-120 of SEQ ID NO: 1.-   27. The composition according to 25, wherein said interhelical loop    region of SNAP-25 comprises at most 35 residues from amino acids    85-120 of SEQ ID NO: 1.-   28. The composition according to 25, wherein said interhelical loop    region of SNAP-25 comprises an amino acid sequence selected from the    group consisting SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ    ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134.-   29. The composition according to 1, wherein said membrane targeting    domain comprises the amino acid motif QPXRV, where X is any amino    acid.-   30. The composition according to 29, wherein said motif comprises an    amino acid sequence selected from the group consisting SEQ ID NO:    135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139,    SEQ ID NO: 140, SEQ ID NO: 141 and SEQ ID NO: 142.-   31. The composition according to 1, wherein said membrane targeting    domain comprises the amino-terminal α-helix region of SNAP-25.-   32. The composition according to 31, wherein said amino-terminal    α-helix region of SNAP-25 comprises at least 5 residues from amino    acids 1-84 of SEQ ID NO: 1-   33. The composition according to 31, wherein said amino-terminal    α-helix region of SNAP-25 comprises at most 80 residues from amino    acids 1-84 of SEQ ID NO: 1-   34. The composition according to 1, wherein said membrane targeting    domain comprises the carboxy-terminal α-helix region of SNAP-25.-   35. The composition according to 34, wherein said carboxy-terminal    α-helix region of SNAP-25 comprises at least 5 residues from amino    acids 121-206 of SEQ ID NO: 1.-   36. The composition according to 34, wherein said carboxy-terminal    α-helix region of SNAP-25 comprises at most 85 residues from amino    acids 121-206 of SEQ ID NO: 1.-   37. The composition according to 1, wherein said substrate comprises    a Clostridial toxin recognition sequence selected from the group    consisting of a BoNT/A recognition sequence including a cleavage    site, a BoNT/B recognition sequence including a cleavage site, a    BoNT/C1 recognition sequence including a cleavage site, a BoNT/D    recognition sequence including a cleavage site, a BoNT/E recognition    sequence including a cleavage site, a BoNT/F recognition sequence    including a cleavage site, a BoNT/G recognition sequence including a    cleavage site and a TeNT recognition sequence including a cleavage    site.-   38. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Gln-Arg.-   39. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Gln-Phe.-   40. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Arg-Ala.

41. The composition according to 1, wherein said Clostridial toxinrecognition sequence comprises at least six consecutive residues ofSyntaxin, said six consecutive residues comprising Lys-Ala.

-   42. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Lys-Leu.-   43. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Arg-Ile.-   44. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Gln-Lys.-   45. The composition according to 1, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Ala-Ala.-   46. A composition comprising a cell population, said population    comprising cells containing an exogenous BoNT/A substrate capable of    being localized to the plasma membrane of said cells; wherein said    cells are capable of BoNT/A intoxication; wherein said exogenous    BoNT/A substrate is comprised of a fluorescent member, a membrane    targeting domain and a BoNT/A recognition sequence comprising a    cleavage site, where the cleavage site intervenes between said    fluorescent member and said membrane localization domain; and    wherein greater than 50% of said cell population comprises said    cells containing said exogenous BoNT/A substrate.-   47. A composition comprising a cell population, said population    comprising cells containing an exogenous BoNT/E substrate capable of    being localized to the plasma membrane of said cells; wherein said    cells are capable of BoNT/E intoxication; wherein said exogenous    BoNT/E substrate is comprised of a fluorescent member, a membrane    targeting domain and a BoNT/E recognition sequence comprising a    cleavage site, where the cleavage site intervenes between said    fluorescent member and said membrane localization domain; and    wherein greater than 50% of said cell population comprises said    cells containing said exogenous BoNT/A substrate.-   48. A method of determining Clostridial toxin activity, said method    comprising the steps of: a. contacting with a sample a cell    population, said population comprising cells containing an exogenous    Clostridial toxin substrate capable of being localized to the plasma    membrane of said cells wherein said cells are capable of Clostridial    toxin intoxication; wherein said exogenous Clostridial toxin    substrate comprises a fluorescent member, a membrane targeting    domain and a Clostridial toxin recognition sequence comprising a    cleavage site, where the cleavage site intervenes between said    fluorescent member and said membrane localization domain; and    wherein greater than 50% of said cell population comprises said    cells containing said exogenous Clostridial toxin substrate; b.    exciting said fluorescent member; and c. determining the    fluorescence of said contacted cell population relative to a control    cell population, where a difference in fluorescence of said    contacted cell population as compared to said control cell    population is indicative of Clostridial toxin activity.-   49. The method according to 48, wherein said sample is selected from    the group consisting of a purified Clostridial toxin, a partially    purified Clostridial toxin or unpurified Clostridial toxin.-   50. The method according to 48, wherein said sample is selected from    the group consisting of a purified Clostridial toxin light chain, a    partially purified Clostridial toxin light chain or unpurified    Clostridial toxin light chain.-   51. The method according to 48, wherein said sample is selected from    the group consisting of a bulk Clostridial toxin, a formulated    Clostridial toxin, a cosmetics Clostridial toxin formulation or a    clinical Clostridial toxin formulation.-   52. The method according to 48, wherein said sample is selected from    the group consisting of a recombinant Clostridial toxin and a    recombinant Clostridial toxin light chain.-   53. The method according to 48, wherein said sample is selected from    the group consisting of a raw food, a cooked food, a partially    cooked food or a processed food.-   54. The method according to 48, wherein said sample is a sample    taken from a mammal.-   55. The method according to 54, wherein said mammalian sample is    selected from the group consisting of a tissue, a saliva, an    excretion or a feces.-   56. The method according to 48, wherein said cell transiently    contains said exogenous Clostridial toxin substrate.-   57. The method according to 48, wherein said cell stably contains    said exogenous Clostridial toxin substrate.-   58. The method according to 48, wherein said substrate is expressed    from a nucleic acid molecule.-   59. The method according to 58, wherein said nucleic acid molecule    comprises a viral expression construct encoding a Clostridial toxin    substrate.-   60. The method according to 48, wherein said toxin substrate is    introduced into said cell using a protein delivery method.-   61. The method according to 48, wherein said cell is a neuronal    cell.-   62. The method according to 61, wherein said neuronal cell is    selected from the group consisting of a primary neuronal cell, an    immortalized neuronal cell and a transformed neuronal cell.-   63. The method according to 61, wherein said neuronal cell is    selected from the group consisting of a neuroblastoma cell, a    neuronal hybrid cell, a spinal cord cell, a central nervous system    cell, a cerebral cortex cell, a dorsal root ganglion cell, a    hippocampal cell and a pheochromocytoma cell.-   64. The method according to 48, wherein said cell is a non-neuronal    cell.-   65. The method according to 64, wherein said non-neuronal cell is    selected from the group consisting of a primary neuronal cell, an    immortalized neuronal cell and a transformed neuronal cell.-   66. The method according to 64, wherein said non-neuronal cell is    selected from the group consisting of an anterior pituitary cell, an    adrenal cell, a pancreatic cell, an ovarian cell, a kidney cell, a    stomach cell, a blood cell, an epithelial cell, a fibroblast, a    thyroid cell, a chondrocyte, a muscle cell, a hepatocyte, a    glandular cell.-   67. The method according to 48, wherein said cell is sensitive to    said intoxication at Clostridial toxin concentrations of 2 nM and    below.-   68. The method according to 48, wherein said cell is sensitive to    said intoxication at Clostridial toxin concentrations of 0.1 nM and    below.-   69. The method according to 48, wherein said cell is sensitive to    said intoxication at Clostridial toxin concentrations of 0.02 nM and    below.-   70. The composition according to 48, wherein said fluorescent member    is a fluorescent protein.-   71. The composition according to 70, wherein said fluorescent    protein is selected from the group consisting of a green fluorescent    protein, a blue fluorescent protein, a cyan fluorescent protein, a    yellow fluorescent protein and a red fluorescent protein.-   72. The composition according to 48, wherein said fluorescent    protein is a fluorophore binding protein.-   73. The composition according to 72, wherein said fluorophore    binding protein is selected from the group consisting of a    tetracysteine peptide, an AGT and a dehalogenase.-   74. The composition according to 73, wherein said tetracysteine    peptide binds to a fluorophore selected from the group consisting of    a nonfluorescent biarsenical derivitive of fluorescein and a    nonfluorescent biarsenical derivitive of resorufin.-   75. The composition according to 73, wherein said AGT binds to a    fluorophore selected from the group consisting of a para-benzyl    guanine diethylaminocoumarin, a para-benzyl guanine    diacetylfluorescein, a para-benzyl guanine dyomic DY-505-05, a    para-benzyl guanine ATTO 488, a para-benzyl guanine ATTO 532, a    para-benzyl guanine dyomic DY-547, a para-benzyl guanine    tetramethylrhodamine, a para-benzyl guanine ATTO 600, a para-benzyl    guanine dyomic DY-632, a para-benzyl guanine dyomic DY-647, a    para-benzyl guanine dyomic DY-732 and a para-benzyl guanine dyomic    DY-747.-   76. The composition according to 73, wherein said dehalogenase binds    to a fluorophore selected from the group consisting of a HaloTag    Coumarian, a HaloTag diAcFAM and a HaloTag TMR.-   77. The composition according to 48, wherein said membrane targeting    domain comprises a interhelical loop region of SNAP-25.-   78. The composition according to 77, wherein said interhelical loop    region of SNAP-25 comprises at least 5 residues from amino acids    85-120 of SEQ ID NO: 1.-   79. The composition according to 77, wherein said interhelical loop    region of SNAP-25 comprises at most 35 residues from amino acids    85-120 of SEQ ID NO: 1.-   80. The composition according to 77, wherein said interhelical loop    region of SNAP-25 comprises an amino acid sequence selected from the    group consisting SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ    ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134.-   81. The composition according to 48, wherein said membrane targeting    domain comprises the amino acid motif QPXRV, where X is any amino    acid.-   82. The composition according to 81, wherein said motif comprises an    amino acid sequence selected from the group consisting SEQ ID NO:    135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139,    SEQ ID NO: 140, SEQ ID NO: 141 and SEQ ID NO: 142.-   83. The composition according to 48, wherein said membrane targeting    domain comprises the amino-terminal α-helix region of SNAP-25.-   84. The composition according to 83, wherein said amino-terminal    α-helix region of SNAP-25 comprises at least 5 residues from amino    acids 1-84 of SEQ ID NO: 1-   85. The composition according to 83, wherein said amino-terminal    α-helix region of SNAP-25 comprises at most 80 residues from amino    acids 1-84 of SEQ ID NO: 1-   86. The composition according to 48, wherein said membrane targeting    domain comprises the carboxy-terminal α-helix region of SNAP-25.-   87. The composition according to 86, wherein said carboxy-terminal    α-helix region of SNAP-25 comprises at least 5 residues from amino    acids 121-206 of SEQ ID NO: 1.-   88. The composition according to 86, wherein said carboxy-terminal    α-helix region of SNAP-25 comprises at most 85 residues from amino    acids 121-206 of SEQ ID NO: 1.-   89. The composition according to 48, wherein said substrate    comprises a Clostridial toxin recognition sequence selected from the    group consisting of a BoNT/A recognition sequence including a    cleavage site, a BoNT/B recognition sequence including a cleavage    site, a BoNT/C1 recognition sequence including a cleavage site, a    BoNT/D recognition sequence including a cleavage site, a BoNT/E    recognition sequence including a cleavage site, a BoNT/F recognition    sequence including a cleavage site, a BoNT/G recognition sequence    including a cleavage site and a TeNT recognition sequence including    a cleavage site.-   90. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Gln-Arg.-   91. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Gln-Phe.-   92. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Arg-Ala.-   93. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    Syntaxin, said six consecutive residues comprising Lys-Ala.-   94. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Lys-Leu.-   95. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    SNAP-25, said six consecutive residues comprising Arg-Ile.-   96. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Gln-Lys.-   97. The composition according to 48, wherein said Clostridial toxin    recognition sequence comprises at least six consecutive residues of    VAMP, said six consecutive residues comprising Ala-Ala.-   98. The method according to 48, wherein determining the fluorescence    of said contacted cell relative to a control cell comprises    detecting an increased fluorescence of the cytoplasmic substrate    from the contacted cell as indicative of Clostridial toxin activity.-   99. The method according to 48, wherein determining the fluorescence    of said contacted cell relative to a control cell comprises    detecting a decrease fluorescence of the membrane-localized    substrate from the contacted cell as indicative of Clostridial toxin    activity.-   100. The method according to 48, wherein determining the    fluorescence of said contacted cell relative to a control cell    comprises detecting a shift in fluorescence intensity of the    membrane-localized substrate to the cytoplasmic substrate of said    contact cell as indicative of Clostridial toxin activity.-   101. The method according to 48, wherein determining the    fluorescence of said contacted cell relative to a control cell    comprises detecting a shift in fluorescence intensity of the    membrane-localized substrate to the cytoplasmic substrate of said    contact cell as indicative of Clostridial toxin activity.-   102. A method of determining BoNT/A activity, said method comprising    the steps of: a. contacting with a sample a cell population, said    population comprising cells containing an exogenous BoNT/A substrate    capable of being localized to the plasma membrane of said cells;    wherein said cells are capable of BoNT/A intoxication; wherein said    exogenous BoNT/A substrate comprises a fluorescent member, a    membrane targeting domain and a BoNT/A recognition sequence    comprising a cleavage site, where the cleavage site intervenes    between said fluorescent member and said membrane localization    domain; and wherein greater than 50% of said cell population    comprises said cells containing said exogenous BoNT/A substrate; b.    exciting said fluorescent member; and c. determining the    fluorescence of said contacted cell population relative to a control    cell population, where a difference in fluorescence of said    contacted cell population as compared to said control cell    population is indicative of BoNT/E activity.-   103. A method of determining BoNT/E activity, said method comprising    the steps of: a. contacting with a sample a cell population, said    population comprising cells containing an exogenous BoNT/E substrate    capable of being localized to the plasma membrane of said cells;    wherein said cells are capable of BoNT/E intoxication; wherein said    exogenous BoNT/E substrate comprises a fluorescent member, a    membrane targeting domain and a BoNT/E recognition sequence    comprising a cleavage site, where the cleavage site intervenes    between said fluorescent member and said membrane localization    domain; and wherein greater than 50% of said cell population    comprises said cells containing said exogenous BoNT/E substrate; b.    exciting said fluorescent member; and c. determining the    fluorescence of said contacted cell population relative to a control    cell population, where a difference in fluorescence of said    contacted cell population as compared to said control cell    population is indicative of BoNT/E activity.

The following examples are intended to illustrate but not limit aspectsof the present invention.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofdisclosed embodiments and are in no way intended to limit any of theembodiments disclosed in the present invention.

Example I Construction of Clostridial Toxin Substrates

1. Construction of BoNT/A, BoNT/C1 and BoNT/E SNAP-25 Substrates

1a. Construction of pQBI25/SNAP-25₂₀₆-GFP

To construct pQBI-25/GFP-SNAP-25₂₀₆, a pGEX/SNAP-25₂₀₆ construct wasdigested with BamHI and EcoRI to excise a fragment containing the entireopen reading frame of SNAP-25₂₀₆. The resulting restriction fragment waspurified by the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia,Calif.), and subcloned using a T4 DNA ligase procedure into a pQBI-25C3vector (Qbiogene, Inc., Irvine, Calif.), digested BamHI and EcoRI, toyield pQBI-25/GFP-SNAP-25₂₀₆. The ligation mixture was transformed intochemically competent E. coli TOP10 cells (Invitrogen, Inc, Carlsbad,Calif.) using a heat shock method, plated on 1.5% Luria-Bertani agarplates (pH 7.0) containing 100 μg/mL of Ampicillin, and placed in a 37°C. incubator for overnight growth. Ampicillin-resistant colonies wereanalyzed using an alkaline lysis plasmid mini-preparation procedure andcandidate expression constructs were screened by restrictionendonuclease mapping to determine the presence and orientation of thecorrect insert fragment. Cultures containing the desired expressionconstruct were used to inoculate 1 L baffled flasks containing 200 mL ofLuria-Bertani media containing 100 μg/mL of Ampicillin and placed in a37° C. incubator, shaking at 250 rpm, for overnight growth. Purifiedplasmid DNA corresponding to an expression construct was isolated usingthe QIAGEN Maxi-prep method (QIAGEN, Inc., Valencia, Calif.) andsequenced to verify that the correct expression construct was made(service contract with Sequetech Corp., Mountain View, Calif.). Thiscloning strategy yielded a pQBI-25 expression construct encoding aGFP-SNAP-25₂₀₆.

To construct pQBI-25/SNAP-25₂₀₆-GFP, a nucleic acid fragment encodingthe amino acid region comprising SNAP-25₂₀₆ is amplified frompQBI-25/GFP-SNAP25₂₀₆ DNA using a polymerase chain reaction method andsubcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The resulting pCR2.1/SNAP-25₂₀₆construct is digested with BamHI and EcoRI to excise a fragmentcontaining the entire open reading frame of SNAP-25₂₀₆. The resultingrestriction fragment was purified by the QIAquick Gel Extraction Kit(QIAGEN, Inc., Valencia, Calif.), and subcloned using a T4 DNA ligaseprocedure into a pQBI-25A2 vector (Qbiogene, Inc., Irvine, Calif.),digested BamHI and EcoRI, to yield pQBI-25/SNAP-25₂₀₆-GFP. The ligationmixture was transformed into chemically competent E. coli TOP10 cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and placed in a 37° C. incubator for overnight growth.Ampicillin-resistant colonies were analyzed using an alkaline lysisplasmid mini-preparation procedure and candidate expression constructswere screened by restriction endonuclease mapping to determine thepresence and orientation of the correct insert fragment. Culturescontaining the desired expression construct were used to inoculate 1 Lbaffled flasks containing 200 mL of Luria-Bertani media containing 100μg/mL of Ampicillin and placed in a 37° C. incubator, shaking at 250rpm, for overnight growth. Purified plasmid DNA corresponding to anexpression construct was isolated using the QIAGEN Maxi-prep method(QIAGEN, Inc., Valencia, Calif.) and sequenced to verify that thecorrect expression construct was made (service contract with SequetechCorp., Mountain View, Calif.). This cloning strategy yielded a pQBI-25expression construct encoding a SNAP-25₂₀₆-GFP.

1b. Construction of pQBI25/SNAP-25₁₃₄-GFP

To construct pQBI-25/SNAP-25₂₀₆-GFP, a nucleic acid fragment encodingthe amino acid region comprising SNAP-25₂₀₆ is amplified frompQBI-25/GFP-SNAP25₂₀₆ DNA using a polymerase chain reaction method andsubcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The resulting pCR2.1/SNAP-25₂₀₆construct is digested with BamHI and EcoRI to excise a fragmentcontaining the entire open reading frame of SNAP-25₂₀₆. The resultingrestriction fragment was purified by the QIAquick Gel Extraction Kit(QIAGEN, Inc., Valencia, Calif.), and subcloned using a T4 DNA ligaseprocedure into a pQBI-25A2 vector (Qbiogene, Inc., Irvine, Calif.),digested BamHI and EcoRI, to yield pQBI-25/SNAP-25₂₀₆-GFP. The ligationmixture was transformed into chemically competent E. coli TOP10 cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and placed in a 37° C. incubator for overnight growth.Ampicillin-resistant colonies were analyzed using an alkaline lysisplasmid mini-preparation procedure and candidate expression constructswere screened by restriction endonuclease mapping to determine thepresence and orientation of the correct insert fragment. Culturescontaining the desired expression construct were used to inoculate 1 Lbaffled flasks containing 200 mL of Luria-Bertani media containing 100μg/mL of Ampicillin and placed in a 37° C. incubator, shaking at 250rpm, for overnight growth. Purified plasmid DNA corresponding to anexpression construct was isolated using the QIAGEN Maxi-prep method(QIAGEN, Inc., Valencia, Calif.) and sequenced to verify that thecorrect expression construct was made (service contract with SequetechCorp., Mountain View, Calif.). This cloning strategy yielded a pQBI-25expression construct encoding a SNAP-25₂₀₆-GFP.

1c. Subcellular Localization of SNAP-25-GFP Substrate and CleavageProducts

In order to determine whether a BoNT/A substrate can localize to thecell membrane, we assessed whether a cell expressing a plasmid encodinga SNAP-25-GFP substrate was able to localized the substrate to the cellmembrane. To transiently express a SNAP-25-GFP substrate in a cell line,a suitable density of PC12 cells was plated into the wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 12), and grown in a 37° C. incubator under 5%carbon dioxide until cells reach the desired density (see Table 12). A500 μL transfection solution is prepared by adding 250 μL of OPTI-MEMReduced Serum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 10 μg of apQBI-25/GFP-SNAP-25₂₀₆ construct or 10 μg of a QBI-25/SNAP-25₂₀₆-GFPconstruct. This transfection was incubated at room temperature forapproximately 20 minutes. The media was replaced with freshunsupplemented media and the 500 μL transfection solution was added tothe cells. The cells were then incubated in a 37° C. incubator under 5%carbon dioxide for approximately 6 to 18 hours. Transfection media wasreplaced with 3 mL of fresh media and incubate cells in a 37° C.incubator under 5% carbon dioxide. After 48 hours, cells were fixed withparaformaldehyde and imaged in a confocal microscope as described in,e.g., Ester Fernandez-Salas et al., Plasma membrane localization signalsin the light chain of botulinum neurotoxin, 101(9) Proc. Natl. Acad.Sci. U.S.A. 3208-3213 (2004). Both the GFP-SNAP25₂₀₆ and SNAP25₂₀₆-GFPsubstrates localized to the plasma membrane (see FIG. 4).

In order to determine whether a cell membrane-localized BoNT/A substratecan be susceptible to BoNT/A cleavage, we assessed whether BoNT/Aexposure to a cell containing a membrane-localized SNAP-25-GFP substrateresulted in the cleavage of the substrate. PC12 cells were transientlytransfected with pQBI-25-SNAP25₂₀₆-GFP a plasmid construct that encodesa SNAP25₂₀₆-GFP substrate, and pcDNA3.1-LC/A, a plasmid construct thatencodes the light chain of BoNT/A as described above in Example I, 1b.Observation of living cells 24 to 48 hours post-transfection using afluorescence inverted microscope indicated that PC12 cells co-expressingSNAP25₂₀₆-GFP and the light chain of BoNT/A resulted in a change influorescent intensity relative to control cells expressing only theSNAP25₂₀₆-GFP substrate. The change in fluorescent intensity resultedfrom a reduced level of fluorescence emitted from the cells, as well asa change in subcellular localization of the GFP fluorescence;fluorescence in the cells co-expressing both substrate and enzyme wasobserved in the cytoplasm with accumulation in some areas or cytoplasmicstructures (see FIG. 4). This cytoplasmic accumulation appears torepresent the cleavage product containing the 9 amino acid cleavagefragment from SNAP-25 fused to GFP. These results indicate that there isa distinct fluorescence pattern as well as a different degree offluorescence in cells containing the uncleaved SNAP25₂₀₆-GFP substrateas compared to cells containing the cleavage products of this substrate(SNAP25₁₉₇ and the remaining 9 residue fragment of SNAP-25 fused toGFP).

1d. Construction of pQBI67/SNAP-25₂₀₆-GFP

To construct pQBI-67/SNAP-25₂₀₆-GFP, a nucleic acid fragment encodingthe amino acid region comprising SNAP-25₂₀₆-GFP substrate is amplifiedfrom pQBI-25/SNAP25₂₀₆-GFP DNA using a polymerase chain reaction methodand subcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/SNAP-25₂₀₆-GFP construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the SNAP-25₂₀₆-GFP peptide; and 2) enable thisinsert to be operably-linked to a pQBI-67 vector (Qbiogene, Inc.,Irvine, Calif.). This insert is subcloned using a T4 DNA ligaseprocedure into a pQBI-67 vector that is digested with appropriaterestriction endonucleases to yield pQBI-67/SNAP-25₂₀₆-GFP. The ligationmixture is transformed into chemically competent E. coli BL21 (DE3)cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mLof Ampicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the SNAP-25₂₀₆-GFP operably-linked to the expression elementsof the pQBI-67 vector.

2. Construction of BoNT/B, BoNT/D, BoNT/F, BoNT/G and TeNT VAMPSubstrates

2a. Construction of pQBI25/VAMP-1-GFP

To make a VAMP-1 substrate suitable for methods disclosed in the presentspecification, a pQBI-25/VAMP-1-GFP construct will be made using aSplicing by Overlapping ends polymerase chain reaction (SOE-PCR)procedure, see, e.g., R. M. Horton et al., Engineering hybrid geneswithout the use of restriction enzymes: gene splicing by overlappingextension, 77(1) Gene 61-68 (1989); and R. M. Horton, PCR-mediatedrecombination and mutagenesis. SOEing together tailor-made genes, 3(2)Mol. Biotechnol. 93-99 (1995). A nucleic acid fragment comprising aregion encoding amino acids 85 to 120 of SNAP-25 (SEQ ID NO: 1) will beoperably-linked by SOE-PCR to a VAMP-1 sequence comprising a regionencoding amino acids 49-92 of SEQ ID NO: 28 and subcloned into a pCR2.1vector using the TOPO® TA cloning method (Invitrogen, Inc, Carlsbad,Calif.). The forward and reverse oligonucleotide primers used for thesereaction are designed to include unique restriction enzyme sites usefulfor subsequent subcloning steps. The resulting pCR2./VAMP-1 construct isdigested with restriction enzymes that 1) excise the insert containingthe entire open reading frame encoding amino acids 85-120 of SNAP-25(SEQ ID NO: 1) and amino acids 49-92 of VAMP-1 (SEQ ID NO: 28); and 2)enable this insert to be operably-linked to a pQBI-25A vector (Qbiogene,Inc., Carlsbad, Calif.). The resulting restriction fragment will bepurified by the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia,Calif.), and will be subcloned using a T4 DNA ligase procedure into apQBI-25A vector (Qbiogene, Inc., Irvine, Calif.) to yieldpQBI-25/VAMP-1-GFP. This cloning strategy yielded a pQBI-25 expressionconstruct encoding a SNAP-25 membrane targeting domain comprising aminoacids 85-120 of SNAP-25 (SEQ ID NO: 1), a Clostridial toxin recognitionsequence comprising amino acids 49-92 of VAMP-1 (SEQ ID NO: 28) and aGFP all operably-linked and suitable to detect activity from BoNT/B,BoNT/D, BoNT/F, BoNT/G or TeNT

The subcellular localization of VAMP-1-GFP substrates and their cleavageproducts will be analyzed using the procedures essentially as describedabove in Example I, 1c, with the exception that the pQBI-25/VAMP-1-GFPconstruct described above in Example I, 2a will be used instead ofexpression constructs encoding SNAP-25₂₀₆-GFP. In addition, a suitableexpression construct encoding the light chain of an appropriateClostridial toxin, such as, e.g., the light chain BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT will be used instead of the pcDNA3.1-LC/Aconstruct.

2b. Construction of pQBI25/VAMP-2-GFP

To make a VAMP-2 substrate suitable for methods disclosed in the presentspecification, a pQBI-25/VAMP-2-GFP construct will be made using aSOE-PCR procedure. A nucleic acid fragment comprising a region encodingamino acids 85 to 120 of SNAP-25 (SEQ ID NO: 1) will be operably-linkedby SOE-PCR to a VAMP-2 sequence comprising a region encoding amino acids47-90 of SEQ ID NO: 31 and subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reaction are designedto include unique restriction enzyme sites useful for subsequentsubcloning steps. The resulting pCR2.1/VAMP-2 construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding amino acids 85-120 of SNAP-25 (SEQ ID NO: 1) andamino acids 47-90 of VAMP-2 (SEQ ID NO: 31); and 2) enable this insertto be operably-linked to a pQBI-25A vector (Qbiogene, Inc., Carlsbad,Calif.). The resulting restriction fragment will be purified by theQIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), and willbe subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.) to yield pQBI-25/VAMP-2-GFP. Thiscloning strategy yielded a pQBI-25 expression construct encoding aSNAP-25 membrane targeting domain comprising amino acids 85-120 ofSNAP-25 (SEQ ID NO: 1), a Clostridial toxin recognition sequencecomprising amino acids 47-90 of VAMP-2 (SEQ ID NO: 31) and a GFP alloperably-linked and suitable to detect activity from BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT

The subcellular localization of VAMP-2-GFP substrates and their cleavageproducts will be analyzed using the procedures essentially as describedabove in Example I, 1c, with the exception that the pQBI-25/VAMP-2-GFPconstruct described above in Example I, 2b will be used instead ofexpression constructs encoding SNAP-25₂₀₆-GFP. In addition, a suitableexpression construct encoding the light chain of an appropriateClostridial toxin, such as, e.g., the light chain BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT will be used instead of the pcDNA3.1-LC/Aconstruct.

2c. Construction of pQBI25/VAMP-3-GFP

To make a VAMP-3 substrate suitable for methods disclosed in the presentspecification, a pQBI-25/VAMP-3-GFP construct will be made using aSOE-PCR procedure. A nucleic acid fragment comprising a region encodingamino acids 85 to 120 of SNAP-25 (SEQ ID NO: 1) will be operably-linkedby SOE-PCR to a VAMP-3 sequence comprising a region encoding amino acids34-77 of SEQ ID NO: 33 and subcloned into a pCR2.1 vector using theTOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forwardand reverse oligonucleotide primers used for these reaction are designedto include unique restriction enzyme sites useful for subsequentsubcloning steps. The resulting pCR2.1/VAMP-3 construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding amino acids 85-120 of SNAP-25 (SEQ ID NO: 1) andamino acids 34-77 of VAMP-3 (SEQ ID NO: 33); and 2) enable this insertto be operably-linked to a pQBI-25A vector (Qbiogene, Inc., Carlsbad,Calif.). The resulting restriction fragment will be purified by theQIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, Calif.), and willbe subcloned using a T4 DNA ligase procedure into a pQBI-25A vector(Qbiogene, Inc., Irvine, Calif.) to yield pQBI-25/VAMP-3-GFP. Thiscloning strategy yielded a pQBI-25 expression construct encoding aSNAP-25 membrane targeting domain comprising amino acids 85-120 ofSNAP-25 (SEQ ID NO: 1), a Clostridial toxin recognition sequencecomprising amino acids 34-77 of VAMP-3 (SEQ ID NO: 33) and a GFP alloperably-linked and suitable to detect activity from BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT

The subcellular localization of VAMP-3-GFP substrates and their cleavageproducts will be analyzed using the procedures essentially as describedabove in Example I, 1c, with the exception that the pQBI-25/VAMP-3-GFPconstruct described above in Example I, 2c will be used instead ofexpression constructs encoding SNAP-25₂₀₆-GFP. In addition, a suitableexpression construct encoding the light chain of an appropriateClostridial toxin, such as, e.g., the light chain BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT will be used instead of the pcDNA3.1-LC/Aconstruct.

2d. Construction of pQBI67/VAMP-1-GFP

To construct pQBI-67/VAMP-1-GFP, a nucleic acid fragment encoding theamino acid region comprising the VAMP-1-GFP substrate as described abovein Example I, 2a, is amplified from pQBI-25/VAMP-1-GFP DNA using apolymerase chain reaction method and subcloned into a pCR2.1 vectorusing the TOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.).The forward and reverse oligonucleotide primers used for this reactionare designed to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting pCR2.1/VAMP-1-GFP constructis digested with restriction enzymes that 1) excise the insertcontaining the entire open reading frame encoding the VAMP-1-GFPpeptide; and 2) enable this insert to be operably-linked to a pQBI-67vector (Qbiogene, Inc., Irvine, Calif.). This insert is subcloned usinga T4 DNA ligase procedure into a pQBI-67 vector that is digested withappropriate restriction endonucleases to yield pQBI-67/VAMP-1-GFP. Theligation mixture is transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin, and placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs are identified asAmpicillin resistant colonies. Candidate constructs are isolated usingan alkaline lysis plasmid mini-preparation procedure and analyzed byrestriction endonuclease digest mapping to determine the presence andorientation of the inset. This cloning strategy yields a mammalianexpression construct encoding the VAMP-1-GFP operably-linked to theexpression elements of the pQBI-67 vector.

2e. Construction of pQBI67/VAMP-2-GFP

To construct pQBI-67/VAMP-2-GFP, a nucleic acid fragment encoding theamino acid region comprising the VAMP-2-GFP substrate as described abovein Example I, 2b, is amplified from pQBI-25/VAMP-2-GFP DNA using apolymerase chain reaction method and subcloned into a pCR2.1 vectorusing the TOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.).The forward and reverse oligonucleotide primers used for this reactionare designed to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting pCR2.1/VAMP-2-GFP constructis digested with restriction enzymes that 1) excise the insertcontaining the entire open reading frame encoding the VAMP-2-GFPpeptide; and 2) enable this insert to be operably-linked to a pQBI-67vector (Qbiogene, Inc., Irvine, Calif.). This insert is subcloned usinga T4 DNA ligase procedure into a pQBI-67 vector that is digested withappropriate restriction endonucleases to yield pQBI-67/VAMP-2-GFP. Theligation mixture is transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin, and placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs are identified asAmpicillin resistant colonies. Candidate constructs are isolated usingan alkaline lysis plasmid mini-preparation procedure and analyzed byrestriction endonuclease digest mapping to determine the presence andorientation of the inset. This cloning strategy yields a mammalianexpression construct encoding the VAMP-2-GFP operably-linked to theexpression elements of the pQBI-67 vector.

2f. Construction of pQBI67/VAMP-3-GFP

To construct pQBI-67/VAMP-3-GFP, a nucleic acid fragment encoding theamino acid region comprising the VAMP-3-GFP substrate as described abovein Example I, 2c, is amplified from pQBI-25/VAMP-3-GFP DNA using apolymerase chain reaction method and subcloned into a pCR2.1 vectorusing the TOPO® TA cloning method (Invitrogen, Inc, Carlsbad, Calif.).The forward and reverse oligonucleotide primers used for this reactionare designed to include unique restriction enzyme sites useful forsubsequent subcloning steps. The resulting pCR2.1/VAMP-3-GFP constructis digested with restriction enzymes that 1) excise the insertcontaining the entire open reading frame encoding the VAMP-3-GFPpeptide; and 2) enable this insert to be operably-linked to a pQBI-67vector (Qbiogene, Inc., Irvine, Calif.). This insert is subcloned usinga T4 DNA ligase procedure into a pQBI-67 vector that is digested withappropriate restriction endonucleases to yield pQBI-67/VAMP-3-GFP. Theligation mixture is transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin, and placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs are identified asAmpicillin resistant colonies. Candidate constructs are isolated usingan alkaline lysis plasmid mini-preparation procedure and analyzed byrestriction endonuclease digest mapping to determine the presence andorientation of the inset. This cloning strategy yields a mammalianexpression construct encoding the VAMP-3-GFP operably-linked to theexpression elements of the pQBI-67 vector.

3. Construction of BoNT/C1 Syntaxin Substrates

3a. Construction of pQBI25/Syntaxin-1-GFP

To make a Syntaxin-1 substrate suitable for methods disclosed in thepresent specification, a pQBI-25/Syntaxin-1-GFP construct will be madeusing a SOE-PCR procedure. A nucleic acid fragment comprising a regionencoding amino acids 85 to 120 of SNAP-25 (SEQ ID NO: 1) will beoperably-linked by SOE-PCR to a Syntaxin-1 sequence comprising a regionencoding amino acids 242-264 of SEQ ID NO: 66 and subcloned into apCR2.1 vector using the TOPO® TA cloning method (Invitrogen, Inc,Carlsbad, Calif.). The forward and reverse oligonucleotide primers usedfor these reaction are designed to include unique restriction enzymesites useful for subsequent subcloning steps. The resultingpCR2.1/Syntaxin-1 construct is digested with restriction enzymes that 1)excise the insert containing the entire open reading frame encodingamino acids 85-120 of SNAP-25 (SEQ ID NO: 1) and amino acids 242-264 ofSyntaxin-1 (SEQ ID NO: 66); and 2) enable this insert to beoperably-linked to a pQBI-25A vector (Qbiogene, Inc., Carlsbad, Calif.).The resulting restriction fragment will be purified by the QIAquick GelExtraction Kit (QIAGEN, Inc., Valencia, Calif.), and will be subclonedusing a T4 DNA ligase procedure into a pQBI-25A vector (Qbiogene, Inc.,Irvine, Calif.) to yield pQBI-25/Syntaxin-1-GFP. This cloning strategyyielded a pQBI-25 expression construct encoding a SNAP-25 membranetargeting domain comprising amino acids 85-120 of SNAP-25 (SEQ ID NO:1), a Clostridial toxin recognition sequence comprising amino acids242-264 of Syntaxin-1 (SEQ ID NO: 66) and a GFP all operably-linked andsuitable to detect activity from BoNT/C1

The subcellular localization of Syntaxin-1-GFP substrates and theircleavage products will be analyzed using the procedures essentially asdescribed above in Example I, 1c, with the exception that thepQBI-25/Syntaxin-1-GFP construct described above in Example I, 2c willbe used instead of expression constructs encoding SNAP-25₂₀₆-GFP. Inaddition, a suitable expression construct encoding the light chain of anappropriate Clostridial toxin, such as, e.g., the light chain BoNT/C1will be used instead of the pcDNA3.1-LC/A construct.

3b. Construction of pQBI67/Syntaxin-1-GFP

To construct pQBI-67/Syntaxin-1-GFP, a nucleic acid fragment encodingthe amino acid region comprising the Syntaxin-1-GFP substrate asdescribed above in Example I, 3a, is amplified frompQBI-25/Syntaxin-1-GFP DNA using a polymerase chain reaction method andsubcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/Syntaxin-1-GFP construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the Syntaxin-1-GFP peptide; and 2) enable thisinsert to be operably-linked to a pQBI-67 vector (Qbiogene, Inc.,Irvine, Calif.). This insert is subcloned using a T4 DNA ligaseprocedure into a pQBI-67 vector that is digested with appropriaterestriction endonucleases to yield pQBI-67/Syntaxin-1-GFP. The ligationmixture is transformed into chemically competent E. coli BL21 (DE3)cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mLof Ampicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the Syntaxin-1-GFP operably-linked to the expression elementsof the pQBI-67 vector.

Example II Identification of Cell Lines with High Affinity Uptake forCoNTs

Distinct sensitivities to each of the CoNT serotypes might be expectedbased on the individual receptor systems for each different toxin andtoxin serotype and their differing expression in different cell lines.The presence of a high affinity receptor system in a cell for CoNT canbe characterized by two attributes: a rapid uptake of the neurotoxin bythe cell, and a low neurotoxin concentration needed for cellintoxication. To identify a cell line having a high affinity receptorsystem for a CoNT, we tested cell lines using one of two different invitro cleavage assay, one to determine the amount of toxin required forintoxication, the other to determine the length of time necessary forthe cell to uptake the neurotoxin.

1. Identification of Cell Lines with High Affinity Uptake for BoNT/A

1a. Assay to Determine the BoNT/A Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/A needed to intoxicate a cell, apanel of mammalian cell lines of neuronal origin was screened todetermine the concentration of toxin necessary to cleave endogenouslyexpressed SNAP-25 (see Table 12). A suitable seed density of cells fromeach line was plated into individual wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 12), and grown in a 37° C. incubator under 5%carbon dioxide for approximately 24 hours. BoNT/A (Metabiologics, Inc.,Madison, Wis.) was added at different concentrations (0 nM, 1 nM, 5 nM,12.5 nM, 25 nM, 50 nM) in the culture medium containing the cells forapproximately 8 or approximately 16 hours. Cells were collected in 15 mltubes, washed once with 1 ml of phosphate-buffered saline, pH 7.4, andthen transferred to 1.5 ml microcentrifuge tubes. Cells were lysed in0.5 ml of lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/ml orhigher concentration.

TABLE 12 Culture Conditions for Cell Lines Cell Line Complete CultureMedia Passage Conditions Seed Density (cells/mm²) SK-N-DZ 90% DMEM, ATrypsin/EDTA treatment, 1:4 dilution split every 2-3 day 4.25 × 10³SK-N-F1 90% DMEM, A Trypsin/EDTA treatment, 1:4 dilution spilt twice aweek 4.25 × 10³ SK-N-SH Ham's F12, DMEM or EMEM, B Trypsin/EDTAtreatment, 1:20 dilution split every 4-7 day 4.25 × 10³ SH-SY5Y EMEM andHam's F12 1:1, C Trypsin/EDTA treatment, 1:6 dilution split every 2-3day 4.25 × 10³ SK-N-BE(2) EMEM and Ham's F12 1:1, D Trypsin/EDTAtreatment, 1:6 dilution split every 3 day 4.25 × 10³ BE(2)-C EMEM andHam's F12 1:1, D Trypsin/EDTA treatment, 1:4 dilution split every 2-3day 4.25 × 10³ BE(2)-M17 EMEM and Ham's F12 1:1, D Trypsin/EDTAtreatment, 1:20 dilution split every 4-7 day 4.25 × 10³ Neuro 2a EMEM, ETrypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 × 10³ C1300RPMI 1640, B Trypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25× 10³ NB4 1A3 Ham's F10, F Trypsin/EDTA treatment, 1:3 dilution splitevery 3 day 4.25 × 10³ N1E-115 DMEM, G Trypsin/EDTA treatment, 1:3dilution split every 3 day 4.25 × 10³ NG108-15 DMEM, B 1:4 dilutionsplit every 1-2 days 4.25 × 10³ HCN-1A DMEM, H Trypsin/EDTA treatment,1:3 dilution split every 3 day 4.25 × 10³ HCN-2 DMEM, H Trypsin/EDTAtreatment, 1:3 dilution split every 3 day 4.25 × 10³ TE 189.T DMEM, HTrypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 × 10³ ND8/34DMEM, B Trypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 ×10³ A contains 1.5 g/L sodium bicarbonate, 0.1 mM Non-essential aminoacids (NEAA), 4 mM Glutamine & 10% Fetal Calf serum (FCS) B contains 2mM Glutamine & 10% FCS C contains 1.5 g/L sodium bicarbonate, 0.1 mMNEAA, 4 mM Glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin(P/S) & 10% FCS D contains 0.1 mM NEAA, 4 mM Glutamine, & 10% FCS Econtains 1.5 g/L sodium bicarbonate, 0.1 mM NEAA, 2 mM Glutamine, 1 mMsodium pyruvate & 10% FCS F contains 2 mM Glutamine, 15% Horse Serum &2.5% FCS G contains 4.5 g/L glucose & 10% FCS H contains 4 mM glucose &10% FCS Freeze medium comprises 95% culture medium and 5% DMSO

To detect for the presence of a cleaved BoNT/A substrate, samples wereboiled for 5 min, and 40 μl aliquots were separated by MOPSpolyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Trisprecast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Separated peptides were transferredfrom the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen,Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SDsemi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories,Hercules, Calif.). PVDF membranes were blocked by incubating at roomtemperature for 2 hours in a solution containing 25 mM Tris-BufferedSaline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride),0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovineserum albumin, 5% nonfat dry milk. Blocked membranes were incubated at4° C. for overnight in Tris-Buffered Saline TWEEN-20® (25 mMTris-Buffered Saline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitanmonolaureate) containing a 1:5,000 dilution of rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25₁₉₇ #1, a polyclonal antibody whichis specific for the SNAP25₁₉₇-cleavage product and does not cross-reactwith full-length SNAP25₂₀₆, (Allergan, Inc., generated under contractwith Zymed Laboratories Inc., South San Francisco, Calif.). Primaryantibody probed blots were washed three times for 15 minutes each timein Tris-Buffered Saline TWEEN-20®. Washed membranes were incubated atroom temperature for 2 hours in Tris-Buffered Saline TWEEN-20,containing a 1:20,000 dilution of goat polyclonal anti-rabbitimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) as a secondary antibody. Secondary antibody-probed blots werewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled BoNT/A SNAP25₁₉₇-cleavageproduct was visualized using the ECL Plus™ Western Blot Detection System(Amersham Biosciences, Piscataway, N.J.) and the membrane was imaged andcleavage product quantitated with a Typhoon 9410 Variable Mode Imagerand Imager Analysis software (Amersham Biosciences, Piscataway, N.J.).The choice of pixel size (100 to 200 pixels) and PMT voltage settings(350 to 600, normally 400) depended on the individual blot. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell lines SH-SY5Y,NG108-15, N1E-115, Neuro-2A and SK-N-BE(2) after at least an 8 hourincubation with at least 5 nM BoNT/A, thereby indicating the ability ofBoNT/A to intoxicate these cell lines (see FIG. 5 a).

The mouse neuroblastoma cell line Neuro-2A was further analyzed withlower concentrations of BoNT/A to determine the concentration ofneurotoxin necessary to cleave endogenously expressed SNAP-25. Cellswere grown in poly-D-lysine/Laminin coated 6-well plates as describedabove in Example II, 1a. BoNT/A (Metabiologics, Inc., Madison, Wis.) wasadded at different concentrations (0 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.5nM, 1 nM, 5 nM and 20 nM) in the culture medium containing cells foreither approximately 8 or approximately 16 hours. Toxin treated cellswere harvested and lysed as described above in Example II, 1a. Thepresence of a BoNT/A SNAP25₁₉₇-cleavage product was determined byWestern blot analysis as described above in Example II, 1a. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell line Neuro-2A afterat least a 8 hour incubation with at least 0.5 nM BoNT/A, therebyindicating the ability of BoNT/A to intoxicate these cell lines (seeFIG. 5 c).

1b. Assay to Determine the Time Required by a Cell to Uptake BoNT/A

In order to assess the amount of time needed by a cell line to uptakeBoNT/A, a panel of mammalian cell lines of neuronal origin was screenedto determine the length of toxin exposure necessary to cleaveendogenously expressed SNAP-25. Cells from each line were grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 1a. Approximately 1 nM BoNT/A (Metabiologics, Inc., Madison, Wis.)was added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells werecollected and lysed as described above in Example II, 1a. The presenceof a BoNT/A SNAP25₁₉₇-cleavage product was determined by Western blotanalysis as described above in Example II, 1a. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell lines Neuro-2A,SH-SY5Y, and NG108-15 after at least an 8 hour incubation with 1 nMBoNT/A, thereby indicating the ability of these cell lines to rapidlyuptake BoNT/A (see FIG. 5 b).

2. Identification of Cell Lines with High Affinity Uptake for BoNT/B

2a. Assay to Determine the BoNT/B Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/B needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin will bescreened to determine the concentration of neurotoxin necessary tocleave endogenously expressed VAMP (see Table 12). Cells will be grownin poly-D-lysine/Laminin coated 6-well plates as described above inExample II, 1a. BoNT/B (Metabiologics, Inc., Madison, Wis.) will beadded at different concentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nMand 100 nM) in the culture medium containing cells for eitherapproximately 8 or approximately 16 hours. Cells will be harvested andlysed as described above in Example II, 1a.

To detect for the presence of a cleaved BoNT/B substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception that blocked PVDF membranes will be incubated in a primaryantibody solution containing one of the following antibodies in order todetect a BoNT/B VAMP-cleavage product rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25₁₉₇ #1:1) 1:1000 dilution of mousemonoclonal anti-VAMP-1 antibody clone CI 10.1 (Synaptic Systems,Goettingen, Germany); 2) 1:20,000 dilution of mouse monoclonalanti-VAMP-2 antibody clone CI 69.1 (Synaptic Systems, Goettingen,Germany); or 3) 1:1000 dilution of mouse monoclonal anti-VAMP-3 antibodyclone CI 10.1 (Synaptic Systems, Goettingen, Germany). In addition, asecondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) will be used rather than the goatpolyclonal anti-rabbit IgG-HRP antibody. Detection of a BoNT/BVAMP-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM BoNT/B will indicate the ability ofBoNT/B to intoxicate these cell lines.

2b. Assay to Determine the Time Required by a Cell to Uptake BoNT/B

In order to assess the amount of time needed by a cell line to uptakeBoNT/B, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed VAMP. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 2a. Approximately 1 nM BoNT/B (Metabiologics, Inc., Madison, Wis.)will be added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells willbe harvested and lysed as described above in Example II, 2a. Thepresence of a BoNT/B VAMP-cleavage product will be determined by Westernblot analysis as described above in Example II, 2a. Detection of aBoNT/B VAMP-cleavage product in a cell line after at least an 8 hourincubation with 1 nM BoNT/B will indicate a cell line that can rapidlyuptake BoNT/B.

3. Identification of Cell Lines with High Affinity Uptake for BoNT/C1

3a. Assay to Determine the BoNT/C1 Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/C1 needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin will bescreened to determine the concentration of neurotoxin necessary tocleave endogenously expressed SNAP-25 or endogenously expressed Syntaxin(see Table 12). Cells will be grown in poly-D-lysine/Laminin coated6-well plates as described above in Example II, 1a. BoNT/C1(Metabiologics, Inc., Madison, Wis.) will be added at differentconcentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM and 100 nM) in theculture medium containing cells for either approximately 8 orapproximately 16 hours. Cells will be harvested and lysed as describedabove in Example II, 1a.

To detect for the presence of a cleaved BoNT/C1 substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception: 1) blocked PVDF membranes will be incubated in a primaryantibody solution containing a 1:50,000 dilution of mouse monoclonalanti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals, Lutherville,Md.) rather than the rabbit polyclonal anti-SNAP25 antiserum pAbanti-SNAP25₁₉₇ #1 and a secondary antibody solution containing a1:20,000 dilution of goat polyclonal anti-mouse immunoglobulin G, heavyand light chains (IgG, H+L) antibody conjugated to horseradishperoxidase (HRP; Pierce Biotechnology, Inc., Rockford, Ill.) rather thanthe goat polyclonal anti-rabbit IgG-HRP antibody in order to detect aBoNT/C1 SNAP25₁₉₈-cleavage product; 2) blocked PVDF membranes will beincubated in a primary antibody solution containing a 1:5000 dilution ofmouse monoclonal anti-Syntaxin-1 antibody clone CI 78.2 (SynapticSystems, Goettingen, Germany) rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25₁₉₇ #1 and a secondary antibodysolution containing a 1:20,000 dilution of goat polyclonal anti-mouseimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) rather than the goat polyclonal anti-rabbit IgG-HRP antibody inorder to detect a BoNT/C1 Syntaxin-cleavage product. Detection of aSNAP25₁₉₈-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM BoNT/C1 will indicate the ability ofBoNT/C1 to intoxicate these cell lines. Detection of a Syntaxin-cleavageproduct in a cell line after at least an 8 hours incubation with atleast 20 nM BoNT/C1 will indicate the ability of BoNT/C1 to intoxicatethese cell lines.

3b. Assay to Determine the Time Required by a Cell to Uptake BoNT/C1

In order to assess the amount of time needed by a cell line to uptakeBoNT/C1, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed SNAP-25 or endogenously expressed Syntaxin. Cellswill be grown in poly-D-lysine/Laminin coated 6-well plates as describedabove in Example II, 3a. Approximately 1 nM BoNT/C1 (Metabiologics,Inc., Madison, Wis.) will be added to the culture medium for 10 min, 20min, 30 min, 60 min 2 hours, 4 hours, 6 hours, 8 hours or 16 hours.Toxin treated cells will be harvested and lysed as described above inExample II, 3a. The presence of a BoNT/C1 SNAP25₁₉₈-cleavage product andBoNT/C1 Syntaxin-cleavage product will be determined by Western blotanalysis as described above in Example II, 3a. Detection of a BoNT/C1SNAP25₁₉₈-cleavage product in a cell line after at least an 8 hourincubation with 1 nM BoNT/C1 will indicate a cell line that can rapidlyuptake BoNT/C1. Detection of a BoNT/C1 Syntaxin-cleavage product in acell line after at least an 8 hour incubation with 1 nM BoNT/C1 willindicate a cell line that can rapidly uptake BoNT/C1 .

4. Identification of Cell Lines with High Affinity Uptake for BoNT/D

4a. Assay to Determine the BoNT/D Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/D needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin will bescreened to determine the concentration of neurotoxin necessary tocleave endogenously expressed VAMP (see Table 12). Cells will be grownin poly-D-lysine/Laminin coated 6-well plates as described above inExample II, 1a. BoNT/D (Metabiologics, Inc., Madison, Wis.) will beadded at different concentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nMand 100 nM) in the culture medium containing cells for eitherapproximately 8 or approximately 16 hours. Cells will be harvested andlysed as described above in Example II, 1a.

To detect for the presence of a cleaved BoNT/D substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception that blocked PVDF membranes will be incubated in a primaryantibody solution containing one of the following antibodies in order todetect a BoNT/D VAMP-cleavage product rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25197 #1:1) 1:1000 dilution of mousemonoclonal anti-VAMP-1 antibody clone CI 10.1 (Synaptic Systems,Goettingen, Germany); 2) 1:20,000 dilution of mouse monoclonalanti-VAMP-2 antibody clone CI 69.1 (Synaptic Systems, Goettingen,Germany); or 3) 1:1000 dilution of mouse monoclonal anti-VAMP-3 antibodyclone CI 10.1 (Synaptic Systems, Goettingen, Germany). In addition, asecondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) will be used rather than the goatpolyclonal anti-rabbit IgG-HRP antibody. Detection of a BoNT/DVAMP-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM BoNT/D will indicate the ability ofBoNT/D to intoxicate these cell lines.

4b. Assay to Determine the Time Required by a Cell to Uptake BoNT/D

In order to assess the amount of time needed by a cell line to uptakeBoNT/D, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed VAMP. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 4a. Approximately 1 nM BoNT/D (Metabiologics, Inc., Madison, Wis.)will be added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells willbe harvested and lysed as described above in Example II, 4a. Thepresence of a BoNT/D VAMP-cleavage product will be determined by Westernblot analysis as described above in Example II, 4a. Detection of aBoNT/D VAMP-cleavage product in a cell line after at least an 8 hourincubation with 1 nM BoNT/D will indicate a cell line that can rapidlyuptake BoNT/D.

5. Identification of Cell Lines with High Affinity Uptake for BoNT/E

5a. Assay to Determine the BoNT/E Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/E needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin was screened todetermine the concentration of neurotoxin necessary to cleaveendogenously expressed SNAP-25 (see Table 12). A suitable density ofcells from each line was plated into individual wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 12), and grown in a 37° C. incubator under 5%carbon dioxide for approximately 24 hours. BoNT/E (Metabiologics, Inc.,Madison, Wis.) was added at different concentrations (0 nM, 2 nM or 20nM) in the culture medium containing cells for either approximately 6 orapproximately 16 hours. Cells were collected in 15 ml tubes, washed oncewith 1 ml of phosphate-buffered saline, pH 7.4, and then transferred to1.5 ml microcentrifuge tubes. Cells were lysed in 0.5 ml of lysis buffercontaining 50 mM N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonicacid) (HEPES), pH 6.8, 150 mM sodium chloride, 1.5 mM magnesiumchloride, 1 mM ethylene glycol bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 10% glycerol and 1% (v/v) Triton-X®100 (4-octylphenol polyethoxylate), with rotation for 1 hour at 4° C.Lysed cells were centrifuged at 5000 rpm for 10 min at 4° C. toeliminate debris and the supernatants were transferred to freshsiliconized tubes. Protein concentrations were measured by Bradford'smethod and resuspended in 1×SDS sample buffer at 1 mg/ml or higherconcentration.

To detect for the presence of a cleaved BoNT/E substrate, western blotanalysis was conducted as described above in Example II, 1a, with theexception that blocked PVDF membranes were incubated in a primaryantibody solution containing a 1:50,000 dilution of mouse monoclonalanti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals, Lutherville,Md.) rather than the rabbit polyclonal anti-SNAP25 antiserum pAbanti-SNAP25197 #1 and a secondary antibody solution containing a1:20,000 dilution of goat polyclonal anti-mouse immunoglobulin G, heavyand light chains (IgG, H+L) antibody conjugated to horseradishperoxidase (HRP; Pierce Biotechnology, Inc., Rockford, Ill.) rather thanthe goat polyclonal anti-rabbit IgG-HRP antibody in order to detect aBoNT/E SNAP25₁₈₀-cleavage product. A BoNT/E SNAP25₁₈₀-cleavage productwas detected in the cell lines Neuro-2A, SH-SY5Y, N1E-115, SK-N-BE(2),NG108-15, SK-N-DZ and BE(2)-C after at least a 6 hour incubation with atleast 20 nM BoNT/E, thereby indicating the ability of BoNT/E tointoxicate these cell lines (see FIG. 6 a).

The human neuroblastoma cell line SK-N-DZ was further analyzed withlower concentrations of BoNT/E to determine the concentration ofneurotoxin necessary to cleave endogenously expressed SNAP-25. Cellswere grown in poly-D-lysine/Laminin coated 6-well plates as describedabove in Example II, 5a. BoNT/E (Metabiologics, Inc., Madison, Wis.) wasadded at different concentrations (0 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.5nM, 1 nM, 2 nM and 5 nM) in the culture medium containing cells forapproximately 6 hours. Toxin treated cells were harvested and lysed asdescribed above in Example II,

5a. The presence of a BoNT/E SNAP25₁₈₀-cleavage product was determinedby Western blot analysis as described above in Example II, 5a. A BoNT/ESNAP25₁₈₀-cleavage product was detected in the cell line SK-N-DZ afterat least a 6 hour incubation with at least 0.1 nM BoNT/E, therebyindicating the ability of BoNT/E to intoxicate these cell lines (seeFIG. 6 c).

5b. Assay to Determine the Time Required by a Cell to Uptake BoNT/E

In order to assess the amount of time needed by a cell line to uptakeBoNT/E, a panel of mammalian cell lines of neuronal origin was screenedto determine the length of toxin exposure necessary to cleaveendogenously expressed SNAP-25 (see Table 12). Cells were grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 5a. Approximately 1 nM BoNT/E (Metabiologics, Inc., Madison, Wis.)was added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells wereharvested and lysed as described above in Example II, 5a. The presenceof a BoNT/E SNAP25₁₈₀-cleavage product was determined by Western blotanalysis as described above in Example II, 5a. A BoNT/ESNAP25₁₈₀-cleavage product was detected in the cell lines Neuro-2A,SH-SY5Y, and NG108-15 after at least an 6 hour incubation with 1 nMBoNT/E, thereby indicating the ability of these cell lines to rapidlyuptake BoNT/E (see FIG. 6 b).

6. Identification of Cell Lines with High Affinity Uptake for BoNT/F

6a. Assay to Determine the BoNT/F Concentration Necessary for cellIntoxication

In order to assess the amount of BoNT/F needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin will bescreened to determine the concentration of neurotoxin necessary tocleave endogenously expressed VAMP (see Table 12). Cells will be grownin poly-D-lysine/Laminin coated 6-well plates as described above inExample II, 1a. BoNT/F (Metabiologics, Inc., Madison, Wis.) will beadded at different concentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nMand 100 nM) in the culture medium containing cells for eitherapproximately 8 or approximately 16 hours. Cells will be harvested andlysed as described above in Example II, 1a.

To detect for the presence of a cleaved BoNT/F substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception that blocked PVDF membranes will be incubated in a primaryantibody solution containing one of the following antibodies in order todetect a BoNT/F VAMP-cleavage product rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25197 #1:1) 1:1000 dilution of mousemonoclonal anti-VAMP-1 antibody clone CI 10.1 (Synaptic Systems,Goettingen, Germany); 2) 1:20,000 dilution of mouse monoclonalanti-VAMP-2 antibody clone CI 69.1 (Synaptic Systems, Goettingen,Germany); or 3) 1:1000 dilution of mouse monoclonal anti-VAMP-3 antibodyclone CI 10.1 (Synaptic Systems, Goettingen, Germany). In addition, asecondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) will be used rather than the goatpolyclonal anti-rabbit IgG-HRP antibody. Detection of a BoNT/FVAMP-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM BoNT/F will indicate the ability ofBoNT/F to intoxicate these cell lines.

6b. Assay to Determine the Time Required by a Cell to Uptake BoNT/F

In order to assess the amount of time needed by a cell line to uptakeBoNT/F, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed VAMP. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 6a. Approximately 1 nM BoNT/F (Metabiologics, Inc., Madison, Wis.)will be added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells willbe harvested and lysed as described above in Example II, 6a. Thepresence of a BoNT/F VAMP-cleavage product will be determined by Westernblot analysis as described above in Example II, 6a. Detection of aBoNT/F VAMP-cleavage product in a cell line after at least an 8 hourincubation with 1 nM BoNT/F will indicate a cell line that can rapidlyuptake BoNT/F.

7. Identification of Cell Lines with High Affinity Uptake for BoNT/G

7a. Assay to Determine the BoNT/G Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/G needed to intoxicate a cellline, a panel of mammalian cell lines of neuronal origin will bescreened to determine the concentration of neurotoxin necessary tocleave endogenously expressed VAMP (see Table 12). Cells will be grownin poly-D-lysine/Laminin coated 6-well plates as described above inExample II, 1a. BoNT/G (Metabiologics, Inc., Madison, Wis.) will beadded at different concentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nMand 100 nM) in the culture medium containing cells for eitherapproximately 8 or approximately 16 hours. Cells will be harvested andlysed as described above in Example II, 1a.

To detect for the presence of a cleaved BoNT/G substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception that blocked PVDF membranes will be incubated in a primaryantibody solution containing one of the following antibodies in order todetect a BoNT/G VAMP-cleavage product rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25197 #1:1) 1:1000 dilution of mousemonoclonal anti-VAMP-1 antibody clone CI 10.1 (Synaptic Systems,Goettingen, Germany); 2) 1:20,000 dilution of mouse monoclonalanti-VAMP-2 antibody clone CI 69.1 (Synaptic Systems, Goettingen,Germany); or 3) 1:1000 dilution of mouse monoclonal anti-VAMP-3 antibodyclone CI 10.1 (Synaptic Systems, Goettingen, Germany). In addition, asecondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) will be used rather than the goatpolyclonal anti-rabbit IgG-HRP antibody. Detection of a BoNT/GVAMP-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM BoNT/G will indicate the ability ofBoNT/G to intoxicate these cell lines.

7b. Assay to Determine the Time Required by a Cell to Uptake BoNT/G

In order to assess the amount of time needed by a cell line to uptakeBoNT/G, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed VAMP. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 7a. Approximately 1 nM BoNT/G (Metabiologics, Inc., Madison, Wis.)will be added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells willbe harvested and lysed as described above in Example II, 7a. Thepresence of a BoNT/G VAMP-cleavage product will be determined by Westernblot analysis as described above in Example II, 7a. Detection of aBoNT/G VAMP-cleavage product in a cell line after at least an 8 hourincubation with 1 nM BoNT/G will indicate a cell line that can rapidlyuptake BoNT/G.

8. Identification of Cell Lines with High Affinity Uptake for TeNT

8a. Assay to Determine the TeNT Concentration Necessary for CellIntoxication

In order to assess the amount of TeNT needed to intoxicate a cell line,a panel of mammalian cell lines of neuronal origin will be screened todetermine the concentration of neurotoxin necessary to cleaveendogenously expressed VAMP (see Table 12). Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 1a. TeNT (Metabiologics, Inc., Madison, Wis.) will be added atdifferent concentrations (0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM and 100nM) in the culture medium containing cells for either approximately 8 orapproximately 16 hours. Cells will be harvested and lysed as describedabove in Example II, 1a.

To detect for the presence of a cleaved TeNT substrate, western blotanalysis will be conducted as described above in Example II, 1a, withthe exception that blocked PVDF membranes will be incubated in a primaryantibody solution containing one of the following antibodies in order todetect a TeNT VAMP-cleavage product rather than the rabbit polyclonalanti-SNAP25 antiserum pAb anti-SNAP25197 #1:1) 1:1000 dilution of mousemonoclonal anti-VAMP-1 antibody clone CI 10.1 (Synaptic Systems,Goettingen, Germany); 2) 1:20,000 dilution of mouse monoclonalanti-VAMP-2 antibody clone CI 69.1 (Synaptic Systems, Goettingen,Germany); or 3) 1:1000 dilution of mouse monoclonal anti-VAMP-3 antibodyclone CI 10.1 (Synaptic Systems, Goettingen, Germany). In addition, asecondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-mouse immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) will be used rather than the goatpolyclonal anti-rabbit IgG-HRP antibody. Detection of a TeNTVAMP-cleavage product in a cell line after at least an 8 hoursincubation with at least 20 nM TeNT will indicate the ability of TeNT tointoxicate these cell lines.

8b. Assay to Determine the Time Required by a Cell to Uptake TeNT

In order to assess the amount of time needed by a cell line to uptakeTeNT, a panel of mammalian cell lines of neuronal origin will bescreened to determine the length of toxin exposure necessary to cleaveendogenously expressed VAMP. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 8a. Approximately 1 nM TeNT (Metabiologics, Inc., Madison, Wis.)will be added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells willbe harvested and lysed as described above in Example II, 8a. Thepresence of a TeNT VAMP-cleavage product will be determined by Westernblot analysis as described above in Example II, 8a. Detection of a TeNTVAMP-cleavage product in a cell line after at least an 8 hour incubationwith 1 nM TeNT will indicate a cell line that can rapidly uptake TeNT.

Example III Treatments to Increase Uptake of a Cell for a ClostridialToxin

Cell surface gangliosides are part of the receptor system forClostridial toxins and appear to participate in binding of a toxin toits receptor system. Although toxin binding is not strictly dependent onthe presence of gangliosides, the presence of specific gangliosidesappears to be required for high affinity binding. In particular, CoNTshave been observed to interact in vitro and in vivo withpolysialogangliosides, especially those of the G1b series (GD1a, GD1b,GD3, GQ1b, or GT1b), see, e.g., Jane L. Halpern & Elaine A. Neale,Neurospecific binding, internalization, and retrograde axonal transport,195 Curr. Top. Microbiol. Immunol. 221-241 (1995). Likewise, thedifferentiated state of a cell could influence the expression, or levelof expression of important components of a Clostridial toxin receptorsystem, such as, e.g., a cell-surface receptor. For example, Neuro-2Aand SH-SY5Y cells can be differentiated to acquire a neuronal-likephenotype that may facilitate toxin uptake. To determine whether wecould increase the uptake of a Clostridial toxin by a particular cell,we tested 1) whether a treatment that increased the ganglioside contentof the cell membrane increased uptake of a Clostridial toxin by a cell;and 2) whether changing the state of differentiation of a cell couldincrease uptake of a Clostridial toxin by a cell.

1. Identification of Treatments that Increased Uptake of BoNT/A by aCell

1a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/A bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/A to intoxicate a cell, a Neuro-2A cell line was pre-treated withdifferent gangliosides to determine whether these sugar moieties couldincrease the uptake of BoNT/A by these cells. Neuro-2A cells were platedat a suitable density into individual wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 12), and grown in a 37° C. incubator under 5%carbon dioxide. After approximately 24 hours, the medium was replaced bya serum-free media and 25 μg/mL of one of the following gangliosides wasadded to individual wells: GD1a, GD1b, GD3, GQ1b, or GT1b (AXXORA, LLC,San Diego, Calif.). After an overnight 37° C. incubation period, theganglioside-treated cells were washed three times with 1 ml ofphosphate-buffered saline, pH 7.4 and then incubated at 37° C. with 1%serum media containing different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) of BoNT/A (Metabiologics, Inc., Madison, Wis.) for approximately8 or approximately 16 hours. Cells were collected in 15 ml tubes, washedonce with 1 ml of phosphate-buffered saline, pH 7.4, and thentransferred to 1.5 ml microcentrifuge tubes. Cells were lysed in 0.5 mlof lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/ml orhigher concentration. The presence of a BoNT/A SNAP25₁₉₇-cleavageproduct was determined by Western blot analysis as described above inExample II, 1a. An increase in BoNT/A SNAP25₁₉₇-cleavage product wasdetected in the Neuro-2A cell line treated with the ganglioside GT1b,thereby indicating that GT1b-treatment can increase the uptake of BoNT/Aby Neuro-2A cells (see FIG. 7 a).

1b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/A by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/A to intoxicate a cell, Neuro-2A and SH-SY5Y cells were treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells could result in anincreased uptake of BoNT/A by these cells. Cells were plated at asuitable density into individual wells of 6-well, poly-D-lysine/Laminincoated, tissue culture plates containing 3 mL of a suitable medium (seeTable 12), and grown in a 37° C. incubator under 5% carbon dioxide.After approximately 24 hours, the medium was replaced with either aserum-free culture media or a 10% serum media and one of the followingdifferentiating reagents was added to individual wells: 0.2 unitsNeuraminidase Type V (Sigma-Aldrich, St. Louis, Mo.), in watercontaining 0.2% ALBUMAX II (Invitrogen, Inc., Carlsbad, Calif.); 20 μMAll Trans-Retinoic acid (Sigma-Aldrich, St. Louis, Mo.) in DMSO(Sigma-Aldrich, St. Louis, Mo.); 1 mM N6,2′-O-Dibutyryladenosine3′:5′-cyclic monophosphate sodium salt (db-cAMP) (Sigma-Aldrich, St.Louis, Mo.); 1 μM Ionomycin, calcium salt (Molecular Probes, Eugene,Oreg.) in DMSO (Sigma-Aldrich, St. Louis, Mo.); or 1×N-2 Supplement(Invitrogen, Inc., 17502-048, Carlsbad, Calif.). After a three day 37°C. incubation period, the serum-free media cells and the reagent-treatedcells were washed three times with 1 ml of phosphate-buffered saline, pH7.4 and then incubated at 37° C. with either serum-free media containing2 nM Pure A (BTX-540) toxin (Metabiologics, Inc., Madison, Wis.) forapproximately 18 hours (the growth condition experiments), or 10% serummedia containing 2 nM Pure A (BTX-540) toxin (Metabiologics, Inc.,Madison, Wis.) for approximately 18 hours (the differentiation reagentexperiments). Cells were harvested by trypsin treatment, collected in 15ml tubes, washed once with 1 ml of phosphate-buffered saline, pH 7.4,and then transferred to 1.5 ml microcentrifuge tubes. Cells were lysedin 0.5 ml of lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 to 2 hours at 4° C. Lysed cells were centrifuged at 5000 rpm for10 min at 4° C. to eliminate debris and the supernatants weretransferred to fresh 1.5 mL siliconized tubes. Protein concentrationswere measured by Bradford's method and resuspended in 1×SDS samplebuffer at 1 mg/ml or higher concentration. The presence of a BoNT/ASNAP25₁₉₇-cleavage product was determined by Western blot analysis asdescribed above in Example II, 1a, with the exception that blocked PVDFmembranes were incubated in a primary antibody solution containing a1:50,000 dilution of mouse monoclonal anti-SNAP-25 antibody (SMI-81;Sternberger Monoclonals, Lutherville, Md.) rather than the rabbitpolyclonal anti-SNAP25 antiserum pAb anti-SNAP25197 #1 and a secondaryantibody solution containing a 1:20,000 dilution of goat polyclonalanti-mouse immunoglobulin G, heavy and light chains (IgG, H+L) antibodyconjugated to horseradish peroxidase (HRP; Pierce Biotechnology, Inc.,Rockford, Ill.) rather than the goat polyclonal anti-rabbit IgG-HRPantibody in order to detect both the uncleaved SNAP-25 and the BoNT/ASNAP25₁₉₇-cleavage product. An increase in BoNT/A SNAP25₁₉₇-cleavageproduct was detected in Neuro-2A and SH-SY5Y cells differentiated inserum-free conditions as compared to 10% serum media, thereby indicatingthat serum-free media conditions can increase the uptake of BoNT/A byNeuro-2A and SH-SY5Y cells (see FIG. 7 b). Likewise, an increase inBoNT/A SNAP25₁₉₇-cleavage product was detected in Neuro-2A cells treatedwith all trans retinoic acid, thereby indicating that retinoic-induceddifferentiation of Neuro-2A can increase the uptake of BoNT/A by thesecells (see FIG. 7 b).

2. Identification of Treatments that Increased Uptake of BoNT/B by aCell

2a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/B bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/B to intoxicate a cell, suitable mammalian cells will bepre-treated with different gangliosides to determine whether these sugarmoieties can increase the uptake of BoNT/B by these cells. Cells will begrown in poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells will be incubated with BoNT/B (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 8 or approximately 16hours. Toxin treated cells will be harvested and lysed as describedabove in Example III, 1a. The presence of a BoNT/B VAMP-cleavage productwill be determined by Western blot analysis as described above inExample II, 2a. An increase in BoNT/B VAMP-cleavage product detected inthe cell line treated with a ganglioside will indicate that treatmentwith that ganglioside can increase the uptake of BoNT/B by these cells.

2b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/B by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/B to intoxicate a cell, suitable mammalian cells will be treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of BoNT/B by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing BoNT/B(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing BoNT/B(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of aBoNT/B VAMP-cleavage product will be determined by Western blot analysisas described above in Example II, 2a. An increase in a BoNT/BVAMP-cleavage product detected in cells grown in serum-free media willindicate that treatment with that reagent can increase the uptake ofBoNT/B by these cells. An increase in a BoNT/B VAMP-cleavage productdetected in cells treated with a differentiation reagent will indicatethat treatment with that reagent can increase the uptake of BoNT/B bythese cells.

3. Identification of Treatments that Increased Uptake of BoNT/C1 by aCell

3a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/C1 bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/C1 to intoxicate a cell, suitable mammalian cells will bepre-treated with different gangliosides to determine whether these sugarmoieties can increase the uptake of BoNT/C1 by these cells. Cells willbe grown in poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells will be incubated with BoNT/C1 (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 8 or approximately 16hours. Toxin treated cells will be harvested and lysed as describedabove in Example III, 1a. The presence of a BoNT/C1 SNAP25₁₈₀-cleavageproduct will be determined by Western blot analysis as described abovein Example II, 3a. The presence of a BoNT/C1 Syntaxin-cleavage productwill be determined by Western blot analysis as described above inExample II, 3a. An increase in BoNT/C1 SNAP25₁₈₀-cleavage productdetected in the cell line treated with a ganglioside will indicate thattreatment with that ganglioside can increase the uptake of BoNT/C1 bythese cells. An increase in BoNT/C1 Syntaxin-cleavage product detectedin the cell line treated with a ganglioside will indicate that treatmentwith that ganglioside can increase the uptake of BoNT/C1 by these cells.

3b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/C1 by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/C1 to intoxicate a cell, suitable mammalian cells will betreated with different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of BoNT/C1 by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing BoNT/C1(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing BoNT/C1(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of aBoNT/C1 SNAP25₁₈₀-cleavage product will be determined by Western blotanalysis as described above in Example II, 3a. The presence of a BoNT/C1Syntaxin-cleavage product will be determined by Western blot analysis asdescribed above in Example II, 3a. An increase in a BoNT/C1SNAP25₁₈₀-cleavage product detected in cells grown in serum-free mediawill indicate that treatment with that reagent can increase the uptakeof BoNT/C1 by these cells. An increase in a BoNT/C1 SNAP25₁₈₀-cleavageproduct detected in cells treated with a differentiation reagent willindicate that treatment with that reagent can increase the uptake ofBoNT/C1 by these cells. An increase in a BoNT/C1 Syntaxin-cleavageproduct detected in cells grown in serum-free media will indicate thattreatment with that reagent can increase the uptake of BoNT/C1 by thesecells. An increase in a BoNT/C1 Syntaxin-cleavage product detected incells treated with a differentiation reagent will indicate thattreatment with that reagent can increase the uptake of BoNT/C1 by thesecells.

4. Identification of Treatments that Increased Uptake of BoNT/D by aCell

4a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/D bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/D to intoxicate a cell, suitable mammalian cells will bepre-treated with different gangliosides to determine whether these sugarmoieties can increase the uptake of BoNT/D by these cells. Cells will begrown in poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells will be incubated with BoNT/D (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 8 or approximately 16hours. Toxin treated cells will be harvested and lysed as describedabove in Example III, 1a. The presence of a BoNT/D VAMP-cleavage productwill be determined by Western blot analysis as described above inExample II, 4a. An increase in BoNT/D VAMP-cleavage product detected inthe cell line treated with a ganglioside will indicate that treatmentwith that ganglioside can increase the uptake of BoNT/D by these cells.

4b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/D by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/D to intoxicate a cell, suitable mammalian cells will be treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of BoNT/D by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing BoNT/D(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing BoNT/D(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of aBoNT/D VAMP-cleavage product will be determined by Western blot analysisas described above in Example II, 4a. An increase in a BoNT/DVAMP-cleavage product detected in cells grown in serum-free media willindicate that treatment with that reagent can increase the uptake ofBoNT/D by these cells. An increase in a BoNT/D VAMP-cleavage productdetected in cells treated with a differentiation reagent will indicatethat treatment with that reagent can increase the uptake of BoNT/D bythese cells.

5. Identification of Treatments that Increased Uptake of BoNT/E by aCell

5a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/E bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/E to intoxicate a cell, a Neuro-2A cell line was pre-treated withdifferent gangliosides to determine whether these sugar moieties couldincrease the uptake of BoNT/E by these cells. Neuro-2A cells were grownin poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells were incubated with BoNT/E (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 6 or approximately 16hours. Toxin treated cells were harvested and lysed as described abovein Example II,

5a. The presence of a BoNT/E SNAP25₁₈₀-cleavage product was determinedby Western blot analysis as described above in Example II, 5a. Anincrease in BoNT/E SNAP25₁₈₀-cleavage product was detected in theNeuro-2A cell lines treated with the gangliosides GD3, GD1b and GD1a,thereby indicating that GD3-treatment, GD1b-treatment or GD1a-treatmentcan increase the uptake of BoNT/E by Neuro-2A cells (see FIG. 8 a).

5b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/E by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/E to intoxicate a cell, SH-SY5Y cells were treated withdifferent growth conditions to determine whether differentiation ofthese cells could result in an increased uptake of BoNT/E by thesecells. SH-SY5Y cells were grown in poly-D-lysine/Laminin coated 6-wellplates using serum-free as described above in Example III, 1b. Theserum-free media cells were incubated with BoNT/E (Metabiologics, Inc.,Madison, Wis.) at concentrations of 5 nM and 20 nM for approximately 30minutes, approximately 1 hour, approximately 2 hours, approximately 4hours, approximately 8 hours and approximately 16 hours. Toxin treatedcells were harvested, collected and lysed as described above in ExampleIII, 1b. The presence of a BoNT/E SNAP25₁₈₀-cleavage product wasdetermined by Western blot analysis as described above in Example II,5a. An increase in BoNT/E SNAP25₁₈₀-cleavage product was detected inSH-SY5Y cells differentiated in serum-free conditions as early as 4hours following exposure to toxin, with a maximal signal evident atleast at 8 hours after BoNT/E-treatment, as compared to 10% serum media,thereby indicating that serum-free media conditions can increase theuptake of BoNT/E by SH-SY5Y cells (see FIG. 8 b).

6. Identification of Treatments that Increased Uptake of BoNT/F by aCell

6a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/F bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/F to intoxicate a cell, suitable mammalian cells will bepre-treated with different gangliosides to determine whether these sugarmoieties can increase the uptake of BoNT/F by these cells. Cells will begrown in poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells will be incubated with BoNT/F (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 8 or approximately 16hours. Toxin treated cells will be harvested and lysed as describedabove in Example III, 1a. The presence of a BoNT/F VAMP-cleavage productwill be determined by Western blot analysis as described above inExample II, 6a. An increase in BoNT/F VAMP-cleavage product detected inthe cell line treated with a ganglioside will indicate that treatmentwith that ganglioside can increase the uptake of BoNT/F by these cells.

6b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/F by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/F to intoxicate a cell, suitable mammalian cells will be treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of BoNT/F by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing BoNT/F(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing BoNT/F(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of aBoNT/F VAMP-cleavage product will be determined by Western blot analysisas described above in Example II, 6a. An increase in a BoNT/FVAMP-cleavage product detected in cells grown in serum-free media willindicate that treatment with that reagent can increase the uptake ofBoNT/F by these cells. An increase in a BoNT/F VAMP-cleavage productdetected in cells treated with a differentiation reagent will indicatethat treatment with that reagent can increase the uptake of BoNT/F bythese cells.

7. Identification of Treatments that Increased Uptake of BoNT/G by aCell

7a. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/G bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/G to intoxicate a cell, suitable mammalian cells will bepre-treated with different gangliosides to determine whether these sugarmoieties can increase the uptake of BoNT/G by these cells. Cells will begrown in poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example III, 1a. Theganglioside-treated cells will be incubated with BoNT/G (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 8 or approximately 16hours. Toxin treated cells will be harvested and lysed as describedabove in Example III, 1a. The presence of a BoNT/G VAMP-cleavage productwill be determined by Western blot analysis as described above inExample II, 7a. An increase in BoNT/G VAMP-cleavage product detected inthe cell line treated with a ganglioside will indicate that treatmentwith that ganglioside can increase the uptake of BoNT/G by these cells.

7b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof BoNT/G by a Cell

In order to assess the effect of cellular differentiation on the abilityof BoNT/G to intoxicate a cell, suitable mammalian cells will be treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of BoNT/G by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing BoNT/G(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing BoNT/G(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of aBoNT/G VAMP-cleavage product will be determined by Western blot analysisas described above in Example II, 7a. An increase in a BoNT/GVAMP-cleavage product detected in cells grown in serum-free media willindicate that treatment with that reagent can increase the uptake ofBoNT/G by these cells. An increase in a BoNT/G VAMP-cleavage productdetected in cells treated with a differentiation reagent will indicatethat treatment with that reagent can increase the uptake of BoNT/G bythese cells.

8. Identification of Treatments that Increased Uptake of TeNT by a Cell

8a. Ganglioside Treatment to Increase High Affinity Uptake of TeNT by aCell

In order to assess the effect of ganglioside treatment on the ability ofTeNT to intoxicate a cell, suitable mammalian cells will be pre-treatedwith different gangliosides to determine whether these sugar moietiescan increase the uptake of TeNT by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates and treated with gangliosidesas described above in Example III, 1a. The ganglioside-treated cellswill be incubated with TeNT (Metabiologics, Inc., Madison, Wis.) atdifferent concentrations (0 nM, 12.5 nM, 25 nM, 50 nM) in 1% serum mediafor either approximately 8 or approximately 16 hours. Toxin treatedcells will be harvested and lysed as described above in Example III, 1a.The presence of a TeNT VAMP-cleavage product will be determined byWestern blot analysis as described above in Example II, 8a. An increasein TeNT VAMP-cleavage product detected in the cell line treated with aganglioside will indicate that treatment with that ganglioside canincrease the uptake of TeNT by these cells.

8b. Differentiation Reagent Treatment to Increase High Affinity Uptakeof TeNT by a Cell

In order to assess the effect of cellular differentiation on the abilityof TeNT to intoxicate a cell, suitable mammalian cells will be treatedwith different growth conditions or differentiation reagents todetermine whether differentiation of these cells can result in anincreased uptake of TeNT by these cells. Cells will be grown inpoly-D-lysine/Laminin coated 6-well plates using either serum-free or10% serum media treated with differentiation reagents as described abovein Example III, 1b. After a three day 37° C. incubation period, theserum-free media cells and the reagent-treated cells will be washedthree times with 1 ml of phosphate-buffered saline, pH 7.4 and then willbe incubated at 37° C. with either serum-free media containing TeNT(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thegrowth condition experiments), or 10% serum media containing TeNT(Metabiologics, Inc., Madison, Wis.) for approximately 18 hours (thedifferentiation reagent experiments). Cells were harvested, collectedand lysed as described above in Example III, 1a. The presence of a TeNTVAMP-cleavage product will be determined by Western blot analysis asdescribed above in Example II, 8a. An increase in a TeNT VAMP-cleavageproduct detected in cells grown in serum-free media will indicate thattreatment with that reagent can increase the uptake of TeNT by thesecells. An increase in a TeNT VAMP-cleavage product detected in cellstreated with a differentiation reagent will indicate that treatment withthat reagent can increase the uptake of TeNT by these cells.

Example IV Generation of Cells Transiently Containing a ClostridialToxin Substrate

1. Generation of Cells Containing a BoNT/A, BoNT/C1 or BoNT/E SNAP-25Substrate by Adenorviral Transduction

1a. Construction of pAd-DEST/SNAP-25₂₀₆-GFP

To make a pAd-DEST/SNAP-25₂₀₆-GFP construct, a nucleic acid fragmentencoding the amino acid region comprising SNAP-25₂₀₆-GFP of is amplifiedfrom pQBI-25/SNAP25₂₀₆-GFP DNA (see Example I, 1a) using a polymerasechain reaction method and subcloned into a pCR2.1 vector using the TOPO®TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forward andreverse oligonucleotide primers used for this reaction are designed toinclude unique restriction enzyme sites useful for subsequent subcloningsteps. The resulting pCR2.1/SNAP-25₂₀₆-GFP construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the SNAP-25₂₀₆-GFP peptide; and 2) enable thisinsert to be operably-linked to a pAd-DEST vector (Invitrogen, Inc.,Carlsbad, Calif.). This insert is subcloned using a T4 DNA ligaseprocedure into a pAd-DEST vector that is digested with appropriaterestriction endonucleases to yield pAd-DEST/SNAP-25₂₀₆-GFP. The ligationmixture is transformed into chemically competent E. coli BL21 (DE3)cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method,plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mLof Ampicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the SNAP-25₂₀₆-GFP operably-linked to the expression elementsof the pAd-DEST vector.

1b. Production of an Adenoviral Stock Containing pAd-DEST/SNAP-25-GFP

To produce an adenoviral stock containing an expression constructencoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate, such as,e.g., pAd-DEST/SNAP-25₂₀₆-GFP, about 5×10⁵ 293A cells are plated in a 35mm tissue culture dish containing 3 mL of complete Dulbecco's ModifiedEagle Media (DMEM), supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 5×10⁵ cells/ml (6-16 hours). One theday of transfection, replace complete, supplemented DMEM media with 2 mLof OPTI-MEM Reduced Serum Medium. A 500 μL transfection solution isprepared by adding 250 μL of OPTI-MEM Reduced Serum Medium containing 15μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated atroom temperature for 5 minutes to 250 μL of OPTI-MEM Reduced SerumMedium containing 5 μg of the linearized expression construct encoding aBoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate, such as, e.g.,pAd-DEST/SNAP-25₂₀₆-GFP. To linearized a pAd-DEST/SNAP-25-GFP construct,5 μg of a pAd-DEST/SNAP-25-GFP construct is digested with PacI (NewEngland Biolabs, Beverly, Mass.). The linearized plasmid is purifiedusing QIAquick kit procedure (QIAGEN, Inc., Valencia, Calif.) and isresuspended in TE Buffer. This transfection is incubated at roomtemperature for approximately 20 minutes. The 500 μL transfectionsolution is then added to the 293A cells and the cells are incubated ina 37° C. incubator under 5% carbon dioxide for approximately 16 hours.Transfection media is replaced with 3 mL of fresh complete, supplementedDMEM and cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 24 hours. The cells are trypsinized and thecontents of each well are transferred to a sterile 10 cm tissue cultureplate containing 10 mL of complete, supplemented DMEM. Replace the oldmedia with fresh complete, supplemented DMEM every 2 or 3 days untilvisible regions of cytopathic effect are observed (typically 7-10 days).Replenish the old culture media with fresh complete, supplemented DMEMand allow the infections to proceed until approximately 80% cytopathiceffect is observed (typically 10-13 days post transfection). Theadenovirus-containing cells are harvested by detaching the cells usingthe culture media and scraping cells from the culture plate. Detachedcells and media are transferred to a 15 mL tube. The harvested cells arelysed using one freeze-thaw round consisting of −80° C. for 30 minutesthen 37° C. for 15 minutes. The cell lysate is centrifuged (5,000×g at20° C. for 15 minutes) to pellet the cellular debris. The clarifiedsupernatant containing the adenoviral particles is transferred to 2 mLcryovials in 1 mL aliquots and should contain approximately 1×10⁷ to 10⁸pfu of adenoviral particles. Aliquots can be stored at −80° C. untilneeded.

1c. Amplification of an Adenoviral Stock Containing pAd-DEST/SNAP-25-GFP

To amplified to the adenoviral stock containing an expression constructencoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate, such as,e.g., pAd-DEST/SNAP-25₂₀₆-GFP, about 3×10⁶ 293A cells are plated in a100 mm culture dish containing in 10 mL of complete Dulbecco's ModifiedEagle Media (DMEM), supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 80-90% confluency (6-16 hours). Thecells are inoculated cells with 100 μL of adenoviral stock and incubatedfor approximately 48-72 hours in a 37° C. incubator under 5% carbondioxide until the cells round up and are floating or lightly attached tothe culture plate. The adenovirus-containing cells are harvested bydetaching the cells using the culture media and scraping cells from theculture plate. Detached cells and media are transferred to a 15 mL tube.The harvested cells are lysed using three freeze-thaw round consistingof −80° C. for 30 minutes then 37° C. for 15 minutes. The cell lysate iscentrifuged (5,000×g at 20° C. for 15 minutes) to pellet the cellulardebris. The clarified supernatant containing the adenoviral particles istransferred to 2 mL cryovials in 1 mL aliquots and should containapproximately 1×10⁸ to 10⁹ pfu of adenoviral particles. Aliquots can bestored at −80° C. until needed.

1d. Transduction of Cells with an Adenoviral Stock ContainingpAd-DEST/SNAP-25-GFP

To transduce cells with the adenoviral stock containing an expressionconstruct encoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate,such as, e.g., pAd-DEST/SNAP-25₂₀₆-GFP, about 1.5×10⁵ SH-SY5Y cells areplated in a 6-well tissue culture dish containing 3 mL of complete 1:1EMEM and Ham's F12 Media (EMEM:F12), supplemented with 10% fetal bovineserum (FBS), 4 mM glutamine (Invitrogen, Inc, Carlsbad, Calif.), 1%sodium pyruvate (Invitrogen, Inc, Carlsbad, Calif.), 1.5 g/L sodiumbicarbonate, 1× penicillin/streptomycin solution (Invitrogen, Inc,Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37° C. incubatorunder 5% carbon dioxide until the cells reach a density of about 5×10⁵cells/ml (6-16 hours). Cells are inoculated with approximately 4 μL ofthe adenoviral stock (approximately 5×10⁸ pfu/ml) and are incubated forapproximately 24 hours in a 37° C. incubator under 5% carbon dioxide.The tranduction media is replaced with 3 mL of fresh complete,supplemented EMEM:F12 and cells are incubated in a 37° C. incubatorunder 5% carbon dioxide for approximately 24 hours. The transduced cellscan be used to conduct a BoNT/A, BoNT/C1 or BoNT/E activity assay usinga SNAP-25-GFP substrate. For greater details on procedures described inthis example, see ViraPower™ Adenoviral Expression System InstructionManual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002).

2. Generation of Cells Containing a BoNT/A, BoNT/C1 or BoNT/E Substrateby Lentiviral Transduction

2a. Construction of pLenti6Ubc/V5-SNAP-25₂₀₆-GFP

To make a pLenti6Ubc/V5-SNAP-25₂₀₆-GFP construct, a nucleic acidfragment encoding the amino acid region comprising a BoNT/A, BoNT/C1 orBoNT/E SNAP-25-GFP substrate is amplified from, e.g.,pQBI-25/SNAP25₂₀₆-GFP DNA (see Example I, 1a) using a polymerase chainreaction method and subcloned into a pCR2.1 vector using the TOPO® TAcloning method (Invitrogen, Inc, Carlsbad, Calif.). The forward andreverse oligonucleotide primers used for this reaction are designed toinclude unique restriction enzyme sites useful for subsequent subcloningsteps. The resulting pCR2.1/SNAP-25₂₀₆-GFP construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the SNAP-25₂₀₆-GFP peptide; and 2) enable thisinsert to be operably-linked to a pLenti6Ubc/V5 vector (Invitrogen,Inc., Carlsbad, Calif.). This insert is subcloned using a T4 DNA ligaseprocedure into a pLenti6Ubc/V5 vector that is digested with appropriaterestriction endonucleases to yield pLenti6Ubc/V5-SNAP-25₂₀₆-GFP. Theligation mixture is transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin, and placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs are identified asAmpicillin resistant colonies. Candidate constructs are isolated usingan alkaline lysis plasmid mini-preparation procedure and analyzed byrestriction endonuclease digest mapping to determine the presence andorientation of the inset. This cloning strategy yields a mammalianexpression construct encoding the SNAP-25₂₀₆-GFP operably-linked to theexpression elements of the pLenti6Ubc/V5 vector an amino-terminal V5peptide.

2b. Production of a Lentiviral Stock ContainingpLenti6Ubc/V5-SNAP-25-GFP

To produce a lentiviral stock containing pLenti6Ubc/V5-SNAP-25-GFP, a3.0 mL transfection solution is prepared by adding 1.5 mL of OPTI-MEMReduced Serum Medium containing 36 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 1.5 mLof OPTI-MEM Reduced Serum Medium containing 3 μg of an expressionconstruct encoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25 substrate, suchas, e.g., pLenti6Ubc/V5-SNAP-25₂₀₆-GFP and 9 μg of ViraPower™ PacakagingMix. After an approximately 20 minute incubation at room temperature,the DNA-lipid complexes are added to a 10 cm tissue culture platecontaining 5 mL OPTI-MEM Reduced Serum Medium. A 5 mL cell suspensioncontaining approximately 6×10⁶ 293A cells are then added to DNA-lipidcomplex media and grown in a 37° C. incubator under 5% carbon dioxideovernight. Transfection media is replaced with 10 mL of completeDulbecco's Modified Eagle Media (DMEM), supplemented with 10% fetalbovine serum (FBS), 2 mM glutamine (Invitrogen, Inc, Carlsbad, Calif.),1 mM sodium pyruvate (Invitrogen, Inc, Carlsbad, Calif.), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide forapproximately 24-48 hours. The lentiovirus-containing cells areharvested by detaching the cells using the culture media and scrapingcells from the culture plate. Detached cells and media are transferredto a 15 mL tube and centrifuged (5,000×g at 20° C. for 15 minutes) topellet the cellular debris. The clarified supernatant containing thelentiviral particles is transferred to 2 mL cryovials in 1 mL aliquotsand should contain approximately 5×10⁵ to 2×10⁷ tu/mL of lentiviralparticles. Aliquots can be stored at −80° C. until needed.

2c. Transduction of Cells with an Lentiviral Stock Containing apLenti6Ubc/V5-SNAP-25-GFP

To transduce cells with the lentiviral stock containingpLenti6Ubc/V5-SNAP-25-GFP, about 1.5×10⁵ SH-SY5Y cells are plated in a6-well tissue culture dish containing 3 mL of complete 1:1 EMEM andHam's F12 Media (EMEM:F12), supplemented with 10% fetal bovine serum(FBS), 4 mM glutamine

(Invitrogen, Inc, Carlsbad, Calif.), 1% sodium pyruvate (Invitrogen,Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate, 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 5×10⁵ cells/ml (6-16 hours). Cellsare inoculated with the lentiviral stock containing an expressionconstruct encoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25 substrate, suchas, e.g., pLenti6Ubc/V5-SNAP-25₂₀₆-GFP (see Example I, 1), using asuitable multiplicity of infection and are incubated for approximately16-24 hours in a 37° C. incubator under 5% carbon dioxide. Thetranduction media is replaced with 3 mL of fresh complete, supplementedEMEM:F12 and cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 24-48 hours. The transduced cells can be usedto conduct a BoNT/A, BoNT/C1 or BoNT/E activity assay using aSNAP-25-GFP substrate. For greater details on procedures described inthis example, see ViraPower™ Lentiviral Expression System InstructionManual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003).

3. Generation of Cells Containing a BoNT/A, BoNT/C1 or BoNT/E Substrateby Protein Transformation

3a. Expression of a SNAP-25-GFP Substrate Using a Bacterial Cell Line

To express a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate usingbacteria, an expression construct encoding a BoNT/A, BoNT/C1 or BoNT/ESNAP-25-GFP substrate, such as, e.g., pQBI-67/SNAP-25₂₀₆-GFP asdescribed above in Example I, 1d, is introduced into chemicallycompetent E. coli BL21 (DE3) cells (Invitrogen, Inc, Carlsbad, Calif.)using a heat-shock transformation protocol. The heat-shock reaction isthen plated onto 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin and placed in a 37° C. incubator for overnightgrowth. A single Ampicillin-resistant colony of transformed E. colicontaining pQBI-67/SNAP-25₂₀₆-GFP is used to inoculate a 15 mL test tubecontaining 3.0 mL Luria-Bertani media, (pH 7.0) containing 100 μg/mL ofAmpicillin which is then placed in a 37° C. incubator, shaking at 250rpm, for overnight growth. The resulting overnight starter culture isused to inoculate a 1.0 L baffled flask containing 100 mL Luria-Bertanimedia, (pH 7.0) containing 100 μg/mL of Ampicillin at a dilution of1:1000. This culture is grown in a 32° C. incubator shaking at 250 rpmfor approximately 6.5 hours until mid-log phase is reached (OD₆₀₀ ofabout 0.6-0.8). Protein expression is then induced by adding 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) and the culture is placed ina 32° C. incubator shaking at 250 rpm for overnight expression. Cellsare harvested by centrifugation (4,000 rpm at 4° C. for 20-30 minutes)to pellet the cells. The supernatant is discarded and the cell pellet isused immediately for subsequent steps, or the pellet is stored at −80°C. until needed.

3b. Expression of a SNAP-25-GFP Substrate Using a Mammalian Cell Line

To express a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate using amammalian cell line, about 1.5×10⁵ SH-SY5Y cells are plated in a 35 mmtissue culture dish containing 3 mL of complete 1:1 EMEM and Ham's F12Media (EMEM:F12), supplemented with 10% fetal bovine serum (FBS), 4 mMglutamine (Invitrogen, Inc, Carlsbad, Calif.), 1% sodium pyruvate(Invitrogen, Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate, 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 5×10⁵ cells/ml (6-16 hours). A 500 μLtransfection solution is prepared by adding 250 μL of OPTI-MEM ReducedSerum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 5 μg of an expressionconstruct encoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25-GFP substrate,such as, e.g., pQBI-25/SNAP25₂₀₆-GFP (see Example I, 1a). Thistransfection is incubated at room temperature for approximately 20minutes. The complete, supplemented EMEM:F12 media is replaced with 2 mLof OPTI-MEM Reduced Serum Medium and the 500 μL transfection solution isadded to the SH-SY5Y cells and the cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 16 hours.Transfection media is replaced with 3 mL of fresh complete, supplementedEMEM:F12 and cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 48 hours. Cells are harvest by rinsing cellsonce with 3.0 mL of 100 mM phosphate-buffered saline, pH 7.4 anddetaching rinsed cells by adding 500 μl of 100 mM phosphate-bufferedsaline, pH 7.4 and scraping cells from the culture plate. Detached cellsare transferred to a 1.5 mL test tube and are pelleted bymicrocentrifugation (10,000×g at 4° C. for 5 minutes). The supernatantis discarded and the cell pellet is used immediately for subsequentsteps, or the pellet is stored at −80° C. until needed.

3c. Purification of a SNAP-25-GFP Substrate

To purify a SNAP-25-GFP substrate, a cell pellet expressing anexpression construct encoding a BoNT/A, BoNT/C1 or BoNT/E SNAP-25substrate, such as, e.g., either a pQBI-67/SNAP-25₂₀₆-GFP or apQBI-25/SNAP-25₂₀₆-GFP construct, is resuspended in 10 mL of Tris-EDTABuffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0), containing 1 mg/mLof lysozyme and the cells are lysed using three freeze-thaw roundsconsisting of −80° C. for 5 minutes then 37° C. for 5 minutes. The celllysate is centrifuged (5,000×g at 4° C. for 15 minutes) to pellet thecellular debris and the supernatant is transferred to a new tubecontaining an equal volume of Column Binding Buffer (4 M ammoniumsulfate). A hydrophobic interaction chromatography (HIC) column isprepared using a 20 mL Econo-Pac column support (Bio-Rad Laboratories,Hercules, Calif.) that is packed with 2.5-5.0 mL of methyl HIC resin(Bio-Rad Laboratories, Hercules, Calif.), which is then equilibrated byrinsing with 5 column volumes of Column Equilibration Buffer (2 Mammonium sulfate). The clarified lysate is applied slowly to theequilibrated column by gravity flow (approximately 0.25-0.3 mL/minute).The column is then washed with 5 column volumes of Column Wash Buffer(1.3 M ammonium sulfate). The SNAP-25₂₀₆-GFP substrate is eluted with20-30 mL of Column Elution Buffer (10 mM TE Buffer) and is collected inapproximately twelve 1 mL fractions. The progress of the SNAP-25₂₀₆-GFPsubstrate sample through the column as well as which elution fractionscontain the sample is monitored using an ultraviolet light from ahand-held transilluminator. The amount of SNAP-25₂₀₆-GFP substratecontained in each elution fraction is determined by a Bradford dyeassay. In this procedure, 20 μL aliquot from each 1.0 mL fraction iscombined with 200 μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories,Hercules, Calif.), diluted 1 to 4 with deionized, distilled water, andthen the intensity of the colorimetric signal is measured using aspectrophotometer. The five fractions with the strongest signal areconsidered to comprise the elution peak and are pooled. Total proteinyield are determined by estimating the total protein concentration ofthe pooled peak elution fractions using bovine gamma globulin as astandard (Bio-Rad Laboratories, Hercules, Calif.). The amount ofSNAP-25₂₀₆-GFP substrate is adjusted to a protein concentration ofapproximately 100 ng/mL.

3d. Protein Transformation of a SNAP-25-GFP Substrate

To transform a SNAP-25-GFP substrate into a mammalian cell line, about1.5×10⁵ SH-SY5Y cells are plated in a 35 mm tissue culture dishcontaining 3 mL of complete 1:1 EMEM and Ham's F12 Media (EMEM:F12),supplemented with 10% fetal bovine serum (FBS), 4 mM glutamine(Invitrogen, Inc, Carlsbad, Calif.), 1% sodium pyruvate (Invitrogen,Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate, 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 5×10⁵ cells/ml (6-16 hours). A 200 μLprotein transfection solution is prepared by adding 100 μL of distilledwater containing 6 μL of Chariot™ protein delivery agent (Active Motif,Carlsbad, Calif.) to 100 μL of 100 mM phosphate-buffered saline, pH 7.4containing 1 μg of a SNAP25-GFP substrate, such as, e.g., SNAP25₂₀₆-GFP,and this solution is incubated at room temperature for approximately 30minutes. After incubation, the cells are washed once by rinsing cellswith 3.0 mL of 100 mM phosphate-buffered saline, pH 7.4. The 200 μLprotein transfection solution is added to the washed cells, followed by400 μL of OPTI-MEM Reduced Serum Medium and the cells are incubated in a37° C. incubator under 5% carbon dioxide for approximately 1 hour. Add 1mL of fresh complete, supplemented EMEM:F12 to the cells and incubate ina 37° C. incubator under 5% carbon dioxide. After 1-2 hours, thetransformed cells can be used to conduct a BoNT/A, BoNT/C1 or BoNT/Eactivity assay.

4. Generation of Cells Containing a BoNT/B, BoNT/D, BoNT/F, BoNT/G orTeNT VAMP Substrate by Adenorviral Transduction

4a. Construction of pAd-DEST/VAMP-GFP

To make a pAd-DEST/VAMP-GFP construct encoding a BoNT/B, BoNT/D, BoNT/F,BoNT/G or TeNT VAMP-GFP substrate, a nucleic acid fragment encoding theamino acid region comprising VAMP-GFP of is amplified from, e.g.,pQBI-25/VAMP-1-GFP DNA, pQBI-25/VAMP-2-GFP DNA or pQBI-25/VAMP-3-GFP DNA(see Examples I, 2a; I, 2b; or I, 2c) using a polymerase chain reactionmethod and subcloned into a pCR2.1 vector using the TOPO® TA cloningmethod (Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/VAMP-GFP construct is digested with restrictionenzymes that 1) excise the insert containing the entire open readingframe encoding the VAMP-GFP peptide; and 2) enable this insert to beoperably-linked to a pAd-DEST vector (Invitrogen, Inc., Carlsbad,Calif.). This insert is subcloned using a T4 DNA ligase procedure into apAd-DEST vector that is digested with appropriate restrictionendonucleases to yield pAd-DEST/VAMP-GFP. The ligation mixture istransformed into chemically competent E. coli BL21 (DE3) cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the VAMP-GFP operably-linked to the expression elements of thepAd-DEST vector.

4b. Production of an Adenoviral Stock Containing pAd-DEST/VAMP-GFP

To produce an adenoviral stock containing an expression constructencoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrate,such as, e.g., pAd-DEST/VAMP-GFP, about 5×10⁵ 293A cells are plated in a35 mm tissue culture dish containing 3 mL of complete Dulbecco'sModified Eagle Media (DMEM), supplemented with 10% fetal bovine serum(FBS), 1× penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad,Calif.) and 1×MEM non-essential amino acids solution (Invitrogen, Inc,Carlsbad, Calif.), and grown in a 37° C. incubator under 5% carbondioxide until the cells reach a density of about 5×10⁵ cells/ml (6-16hours). One the day of transfection, replace complete, supplemented DMEMmedia with 2 mL of OPTI-MEM Reduced Serum Medium. A 500 μL transfectionsolution is prepared by adding 250 μL of OPTI-MEM Reduced Serum Mediumcontaining 15 μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.)incubated at room temperature for 5 minutes to 250 μL of OPTI-MEMReduced Serum Medium containing 5 μg of the linearized expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, such as, e.g., pAd-DEST/VAMP-GFP. To linearized apAd-DEST/VAMP-GFP construct, 5 μg of a pAd-DEST/VAMP-GFP construct isdigested with PacI (New England Biolabs, Beverly, Mass.). The linearizedplasmid is purified using QIAquick kit procedure (QIAGEN, Inc.,Valencia, Calif.) and is resuspended in TE Buffer. This transfection isincubated at room temperature for approximately 20 minutes. The 500 μLtransfection solution is then added to the 293A cells and the cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 16 hours. Transfection media is replaced with 3 mL offresh complete, supplemented DMEM and cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 24 hours. Trypsinizethe cells and transfer the contains of each well to a sterile 10 cmtissue culture plate containing 10 mL of complete, supplemented DMEM.Replace the old media with fresh complete, supplemented DMEM every 2 or3 days until visible regions of cytopathic effect are observed(typically 7-10 days). Replenish the old culture media with freshcomplete, supplemented DMEM and allow the infections to proceed untilapproximately 80% cytopathic effect is observed (typically 10-13 dayspost transfection). The adenovirus-containing cells are harvested bydetaching the cells using the culture media and scraping cells from theculture plate. Detached cells and media are transferred to a 15 mL tube.The harvested cells are lysed using one freeze-thaw round consisting of−80° C. for 30 minutes then 37° C. for 15 minutes. The cell lysate iscentrifuged (5,000×g at 20° C. for 15 minutes) to pellet the cellulardebris. The clarified supernatant containing the adenoviral particles istransferred to 2 mL cryovials in 1 mL aliquots and should containapproximately 1×10⁷ to 10⁸ pfu of adenoviral particles. Aliquots can bestored at −80° C. until needed.

4c. Amplification of an Adenoviral Stock Containing pAd-DEST/VAMP-GFP

To amplified to the adenoviral stock containing an expression constructencoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrate,such as, e.g., pAd-DEST/VAMP-GFP, about 3×10⁶ 293A cells are plated in a100 mm culture dish containing in 10 mL of complete Dulbecco's ModifiedEagle Media (DMEM), supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 80-90% confluency (6-16 hours). Thecells are inoculated cells with 100 μL of adenoviral stock and incubatedfor approximately 48-72 hours in a 37° C. incubator under 5% carbondioxide until the cells round up and are floating or lightly attached tothe culture plate. The adenovirus-containing cells are harvested bydetaching the cells using the culture media and scraping cells from theculture plate. Detached cells and media are transferred to a 15 mL tube.The harvested cells are lysed using three freeze-thaw round consistingof −80° C. for 30 minutes then 37° C. for 15 minutes. The cell lysate iscentrifuged (5,000×g at 20° C. for 15 minutes) to pellet the cellulardebris. The clarified supernatant containing the adenoviral particles istransferred to 2 mL cryovials in 1 mL aliquots and should containapproximately 1×10⁸ to 10⁹ pfu of adenoviral particles. Aliquots can bestored at −80° C. until needed.

4d. Transduction of Cells with an Adenoviral Stock ContainingpAd-DEST/VAMP-GFP

To transduce cells with the adenoviral stock containing an expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, such as, e.g., pAd-DEST/VAMP-GFP, a suitable density (1×10⁵to 1×10⁶) of appropriate cells are plated in a 6-well tissue culturedish containing 3 mL of complete, supplemented culture media and aregrown in a 37° C. incubator under 5% carbon dioxide until the cellsreach a density appropriate for transduction. Cells are inoculated withapproximately 4 μL of the adenoviral stock (approximately 5×10⁸ pfu/ml)and are incubated for approximately 24 hours in a 37° C. incubator under5% carbon dioxide. The tranduction media is replaced with 3 mL of freshcomplete, supplemented media and cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 24 hours. Thetransduced cells can be used to conduct a BoNT/B, BoNT/D, BoNT/F, BoNT/Gor TeNT activity assay using a VAMP-GFP substrate. For greater detailson procedures described in this example, see ViraPower™ AdenoviralExpression System Instruction Manual 25-0543 version A, Invitrogen,Inc., (Jul. 15, 2002).

5. Generation of Cells Containing a BoNT/B, BoNT/D, BoNT/F, BoNT/G orTeNT Substrate by Lentiviral Transduction

5a. Construction of pLenti6Ubc/V5-VAMP-GFP

To make a pLenti6Ubc/V5-VAMP-GFP construct encoding a BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT VAMP-GFP substrate, a nucleic acid fragmentencoding the amino acid region comprising a BoNT/B, BoNT/D, BoNT/F,BoNT/G or TeNT VAMP-GFP substrate is amplified from, e.g., e.g.,pQBI-25/VAMP-1-GFP DNA, pQBI-25/VAMP-2-GFP DNA or pQBI-25/VAMP-3-GFP DNA(see Examples I, 2a; I, 2b; or I, 2c), using a polymerase chain reactionmethod and subcloned into a pCR2.1 vector using the TOPO® TA cloningmethod (Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/VAMP-GFP construct is digested with restrictionenzymes that 1) excise the insert containing the entire open readingframe encoding the VAMP-GFP peptide; and 2) enable this insert to beoperably-linked to a pLenti6Ubc/V5 vector (Invitrogen, Inc., Carlsbad,Calif.). This insert is subcloned using a T4 DNA ligase procedure into apLenti6Ubc/V5 vector that is digested with appropriate restrictionendonucleases to yield pLenti6Ubc/V5-VAMP-GFP. The ligation mixture istransformed into chemically competent E. coli BL21 (DE3) cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the VAMP-GFP operably-linked to the expression elements of thepLenti6Ubc/V5 vector an amino-terminal V5 peptide.

5b. Production of a Lentiviral Stock Containing pLenti6Ubc/V5-VAMP-GFP

To produce a lentiviral stock containing an expression constructencoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrate, a3.0 mL transfection solution is prepared by adding 1.5 mL of OPTI-MEMReduced Serum Medium containing 36 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 1.5 mLof OPTI-MEM Reduced Serum Medium containing 3 μg of an expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, such as, e.g., pLenti6Ubc/V5-VAMP-GFP and 9 μg of ViraPower™Pacakaging Mix. After an approximately 20 minute incubation at roomtemperature, the DNA-lipid complexes are added to a 10 cm tissue cultureplate containing 5 mL OPTI-MEM Reduced Serum Medium. A 5 mL cellsuspension containing approximately 6×10⁶ 293A cells are then added toDNA-lipid complex media and grown in a 37° C. incubator under 5% carbondioxide overnight. Transfection media is replaced with 10 mL of completeDulbecco's Modified Eagle Media (DMEM), supplemented with 10% fetalbovine serum (FBS), 2 mM glutamine (Invitrogen, Inc, Carlsbad, Calif.),1 mM sodium pyruvate (Invitrogen, Inc, Carlsbad, Calif.), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide forapproximately 24-48 hours. The lentiovirus-containing cells areharvested by detaching the cells using the culture media and scrapingcells from the culture plate. Detached cells and media are transferredto a 15 mL tube and centrifuged (5,000×g at 20° C. for 15 minutes) topellet the cellular debris. The clarified supernatant containing thelentiviral particles is transferred to 2 mL cryovials in 1 mL aliquotsand should contain approximately 5×10⁵ to 2×10⁷ tu/mL of lentiviralparticles. Aliquots can be stored at −80° C. until needed.

5c. Transduction of Cells with an Lentiviral Stock Containing apLenti6Ubc/V5-VAMP-GFP

To transduce cells with the lentiviral stock containing an expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, a suitable density (1.5×10⁵ to 1.5×10⁶) of appropriate cellsis plated in a 6-well tissue culture dish containing 3 mL of complete,supplemented culture media and the cells are grown in a 37° C. incubatorunder 5% carbon dioxide until the cells reach a density of about 5×10⁵cells/ml (6-16 hours). Cells are inoculated with the lentiviral stockcontaining an expression construct encoding a BoNT/B, BoNT/D, BoNT/F,BoNT/G or TeNT VAMP-GFP substrate, such as, e.g.,pLenti6Ubc/V5-VAMP-GFP, using a suitable multiplicity of infection andare incubated for approximately 16-24 hours in a 37° C. incubator under5% carbon dioxide. The tranduction media is replaced with 3 mL of freshcomplete, supplemented media and cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 24-48 hours. Thetransduced cells can be used to conduct a BoNT/B, BoNT/D, BoNT/F, BoNT/Gor TeNT activity assay using a VAMP-GFP substrate. For greater detailson procedures described in this example, see ViraPower™ LentiviralExpression System Instruction Manual 25-0501 version E, Invitrogen,Inc., (Dec. 8, 2003).

6. Generation of Cells Containing a BoNT/B, BoNT/D, BoNT/F, BoNT/G orTeNT Substrate by Protein Transformation

6a. Expression of a VAMP-GFP Substrate Using a Bacterial Cell Line

To express a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrateusing bacteria, an expression construct encoding a BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT VAMP-GFP substrate, such as, e.g.,pQBI-67/VAMP-1-GFP, pQBI-67/VAMP-2-GFP or pQBI-67/VAMP-3-GFP asdescribed above in Examples I, 2-d; I, 2e; or I, 2f, is introduced intochemically competent E. coli BL21 (DE3) cells (Invitrogen, Inc,Carlsbad, Calif.) using a heat-shock transformation protocol. Theheat-shock reaction is then plated onto 1.5% Luria-Bertani agar plates(pH 7.0) containing 100 μg/mL of Ampicillin and placed in a 37° C.incubator for overnight growth. A single Ampicillin-resistant colony oftransformed E. coli containing pQBI-67/VAMP-GFP is used to inoculate a15 mL test tube containing 3.0 mL Luria-Bertani media, (pH 7.0)containing 100 μg/mL of Ampicillin which is then placed in a 37° C.incubator, shaking at 250 rpm, for overnight growth. The resultingovernight starter culture is used to inoculate a 1.0 L baffled flaskcontaining 100 mL Luria-Bertani media, (pH 7.0) containing 100 μg/mL ofAmpicillin at a dilution of 1:1000. This culture is grown in a 32° C.incubator shaking at 250 rpm for approximately 6.5 hours until mid-logphase is reached (OD₆₀₀ of about 0.6-0.8). Protein expression is theninduced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) andthe culture is placed in a 32° C. incubator shaking at 250 rpm forovernight expression. Cells are harvested by centrifugation (4,000 rpmat 4° C. for 20-30 minutes) to pellet the cells. The supernatant isdiscarded and the cell pellet is used immediately for subsequent steps,or the pellet is stored at −80° C. until needed.

6b. Expression of a VAMP-GFP Substrate Using a Mammalian Cell Line

To express a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrateusing a mammalian cell line, a suitable density (1.0×10⁵ to 1.0×10⁶) ofappropriate cells is plated in a 35 mm tissue culture dish containing 3mL of complete, supplemented culture media and the cells are grown in a37° C. incubator under 5% carbon dioxide until the cells reach a densityof about 5×10⁵ cells/ml (6-16 hours). A 500 μL transfection solution isprepared by adding 250 μL of OPTI-MEM Reduced Serum Medium containing 15μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated atroom temperature for 5 minutes to 250 μL of OPTI-MEM Reduced SerumMedium containing 5 μg of an expression construct encoding a BoNT/B,BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrate, such as, e.g.,pQBI-25/VAMP-1-GFP, pQBI-25/VAMP-2-GFP or pQBI-25/VAMP-3-GFP (seeExamples I, 2a; I, 2b; or I, 2c). This transfection is incubated at roomtemperature for approximately 20 minutes. The complete, supplementedculture media is replaced with 2 mL of OPTI-MEM Reduced Serum Medium andthe 500 μL transfection solution is added to the cells and the cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 16 hours. Transfection media is replaced with 3 mL offresh complete, supplemented EMEM:F12 and cells are incubated in a 37°C. incubator under 5% carbon dioxide for approximately 48 hours. Cellsare harvest by rinsing cells once with 3.0 mL of 100 mMphosphate-buffered saline, pH 7.4 and detaching rinsed cells by adding500 μl of 100 mM phosphate-buffered saline, pH 7.4 and scraping cellsfrom the culture plate. Detached cells are transferred to a 1.5 mL testtube and are pelleted by microcentrifugation (10,000×g at 4° C. for 5minutes). The supernatant is discarded and the cell pellet is usedimmediately for subsequent steps, or the pellet is stored at −80° C.until needed.

6c. Purification of a VAMP-GFP Substrate

To purify a VAMP-GFP substrate, a cell pellet expressing an expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, such as, e.g., either a pQBI-67/VAMP-1-GFP or apQBI-25/VAMP-1-GFP construct, is resuspended in 10 mL of Tris-EDTABuffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0), containing 1 mg/mLof lysozyme and the cells are lysed using three freeze-thaw roundsconsisting of −80° C. for 5 minutes then 37° C. for 5 minutes. The celllysate is centrifuged (5,000×g at 4° C. for 15 minutes) to pellet thecellular debris and the supernatant is transferred to a new tubecontaining an equal volume of Column Binding Buffer (4 M ammoniumsulfate). A hydrophobic interaction chromatography (HIC) column isprepared using a 20 mL Econo-Pac column support (Bio-Rad Laboratories,Hercules, Calif.) that is packed with 2.5-5.0 mL of methyl HIC resin(Bio-Rad Laboratories, Hercules, Calif.), which is then equilibrated byrinsing with 5 column volumes of Column Equilibration Buffer (2 Mammonium sulfate). The clarified lysate is applied slowly to theequilibrated column by gravity flow (approximately 0.25-0.3 mL/minute).The column is then washed with 5 column volumes of Column Wash Buffer(1.3 M ammonium sulfate). The VAMP-GFP substrate is eluted with 20-30 mLof Column Elution Buffer (10 mM TE Buffer) and is collected inapproximately twelve 1 mL fractions. The progress of the VAMP-GFPsubstrate sample through the column as well as which elution fractionscontain the sample is monitored using an ultraviolet light from ahand-held transilluminator. The amount of VAMP-GFP substrate containedin each elution fraction is determined by a Bradford dye assay. In thisprocedure, 20 μL aliquot from each 1.0 mL fraction is combined with 200μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, Calif.),diluted 1 to 4 with deionized, distilled water, and then the intensityof the colorimetric signal is measured using a spectrophotometer. Thefive fractions with the strongest signal are considered to comprise theelution peak and are pooled. Total protein yield are determined byestimating the total protein concentration of the pooled peak elutionfractions using bovine gamma globulin as a standard (Bio-RadLaboratories, Hercules, Calif.). The amount of VAMP-GFP substrate isadjusted to a protein concentration of approximately 100 ng/mL.

6d. Protein Transformation of a VAMP-GFP Substrate

To transform a VAMP-GFP substrate into a mammalian cell line, a suitabledensity (1.0×10⁵ to 1.0×10⁶) of appropriate cells is plated in a 35 mmtissue culture dish containing 3 mL of complete, supplemented culturemedia and the cells are grown in a 37° C. incubator under 5% carbondioxide until the cells reach a density of about 5×10⁵ cells/ml (6-16hours). A 200 μL protein transfection solution is prepared by adding 100μL of distilled water containing 6 μL of Chariot™ protein delivery agent(Active Motif, Carlsbad, Calif.) to 100 μL of 100 mM phosphate-bufferedsaline, pH 7.4 containing 1 μg of a VAMP-GFP substrate and this solutionis incubated at room temperature for approximately 30 minutes. Afterincubation, the cells are washed once by rinsing cells with 3.0 mL of100 mM phosphate-buffered saline, pH 7.4. The 200 μL proteintransfection solution is added to the washed cells, followed by 400 μLof OPTI-MEM Reduced Serum Medium and the cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 1 hour. Add 1 mL offresh complete, supplemented culture media to the cells and incubate ina 37° C. incubator under 5% carbon dioxide. After 1-2 hours, thetransformed cells can be used to conduct a BoNT/B, BoNT/D, BoNT/F,BoNT/G or TeNT activity assay using a VAMP-GFP substrate.

7. Generation of Cells Containing a BoNT/C1 Syntaxin Substrate byAdenorviral Transduction

7a. Construction of pAd-DEST/Syntaxin-GFP

To make a pAd-DEST/Syntaxin-GFP construct encoding a BoNT/C1Syntaxin-GFP substrate, a nucleic acid fragment encoding the amino acidregion comprising Syntaxin-GFP of is amplified an expression constructencoding a BoNT/C1-GFP substrate, such as, e.g., pQBI-25/Syntaxin-1-GFPDNA (see Example I, 3a) using a polymerase chain reaction method andsubcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/Syntaxin-GFP construct is digested with restrictionenzymes that 1) excise the insert containing the entire open readingframe encoding the Syntaxin-GFP peptide; and 2) enable this insert to beoperably-linked to a pAd-DEST vector (Invitrogen, Inc., Carlsbad,Calif.). This insert is subcloned using a T4 DNA ligase procedure into apAd-DEST vector that is digested with appropriate restrictionendonucleases to yield pAd-DEST/Syntaxin-GFP. The ligation mixture istransformed into chemically competent E. coli BL21 (DE3) cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat shock method, plated on1.5% Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL ofAmpicillin, and placed in a 37° C. incubator for overnight growth.Bacteria containing expression constructs are identified as Ampicillinresistant colonies. Candidate constructs are isolated using an alkalinelysis plasmid mini-preparation procedure and analyzed by restrictionendonuclease digest mapping to determine the presence and orientation ofthe inset. This cloning strategy yields a mammalian expression constructencoding the Syntaxin-GFP operably-linked to the expression elements ofthe pAd-DEST vector.

7b. Production of an Adenoviral Stock Containing pAd-DEST/Syntaxin-GFP

To produce an adenoviral stock containing an expression constructencoding a BoNT/C1 Syntaxin-GFP substrate, about 5×10⁵ 293A cells areplated in a 35 mm tissue culture dish containing 3 mL of completeDulbecco's Modified Eagle Media (DMEM), supplemented with 10% fetalbovine serum (FBS), 1× penicillin/streptomycin solution (Invitrogen,Inc, Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37° C. incubatorunder 5% carbon dioxide until the cells reach a density of about 5×10⁵cells/ml (6-16 hours). One the day of transfection, replace complete,supplemented DMEM media with 2 mL of OPTI-MEM Reduced Serum Medium. A500 μL transfection solution is prepared by adding 250 μL of OPTI-MEMReduced Serum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 5 μg of the linearizedexpression construct encoding a BoNT/C1 Syntaxin-GFP substrate, such as,e.g., pAd-DEST/Syntaxin-GFP. To linearized a pAd-DEST/Syntaxin-GFPconstruct, 5 μg of a pAd-DEST/Syntaxin-GFP construct is digested withPacI (New England Biolabs, Beverly, Mass.). The linearized plasmid ispurified using QIAquick kit procedure (QIAGEN, Inc., Valencia, Calif.)and is resuspended in TE Buffer. This transfection is incubated at roomtemperature for approximately 20 minutes. The 500 μL transfectionsolution is then added to the 293A cells and the cells are incubated ina 37° C. incubator under 5% carbon dioxide for approximately 16 hours.Transfection media is replaced with 3 mL of fresh complete, supplementedDMEM and cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 24 hours. Trypsinize the cells and transferthe contains of each well to a sterile 10 cm tissue culture platecontaining 10 mL of complete, supplemented DMEM. Replace the old mediawith fresh complete, supplemented DMEM every 2 or 3 days until visibleregions of cytopathic effect are observed (typically 7-10 days).Replenish the old culture media with fresh complete, supplemented DMEMand allow the infections to proceed until approximately 80% cytopathiceffect is observed (typically 10-13 days post transfection). Theadenovirus-containing cells are harvested by detaching the cells usingthe culture media and scraping cells from the culture plate. Detachedcells and media are transferred to a 15 mL tube. The harvested cells arelysed using one freeze-thaw round consisting of −80° C. for 30 minutesthen 37° C. for 15 minutes. The cell lysate is centrifuged (5,000×g at20° C. for 15 minutes) to pellet the cellular debris. The clarifiedsupernatant containing the adenoviral particles is transferred to 2 mLcryovials in 1 mL aliquots and should contain approximately 1×10⁷ to 10⁸pfu of adenoviral particles. Aliquots can be stored at −80° C. untilneeded.

7c. Amplification of an Adenoviral Stock ContainingpAd-DEST/Syntaxin-GFP

To amplified to the adenoviral stock containing an expression constructencoding a BoNT/C1 Syntaxin-GFP substrate, such as, e.g.,pAd-DEST/Syntaxin-GFP, about 3×10⁶ 293A cells are plated in a 100 mmculture dish containing in 10 mL of complete Dulbecco's Modified EagleMedia (DMEM), supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 80-90% confluency (6-16 hours). Thecells are inoculated cells with 100 μL of adenoviral stock and incubatedfor approximately 48-72 hours in a 37° C. incubator under 5% carbondioxide until the cells round up and are floating or lightly attached tothe culture plate. The adenovirus-containing cells are harvested bydetaching the cells using the culture media and scraping cells from theculture plate. Detached cells and media are transferred to a 15 mL tube.The harvested cells are lysed using three freeze-thaw round consistingof −80° C. for 30 minutes then 37° C. for 15 minutes. The cell lysate iscentrifuged (5,000×g at 20° C. for 15 minutes) to pellet the cellulardebris. The clarified supernatant containing the adenoviral particles istransferred to 2 mL cryovials in 1 mL aliquots and should containapproximately 1×10⁸ to 10⁹ pfu of adenoviral particles. Aliquots can bestored at −80° C. until needed.

7d. Transduction of Cells with an Adenoviral Stock ContainingpAd-DEST/Syntaxin-GFP

To transduce cells with the adenoviral stock containing an expressionconstruct encoding a BoNT/C1 Syntaxin-GFP substrate, such as, e.g.,pAd-DEST/Syntaxin-GFP, a suitable density (1×10⁵ to 1×10⁶) ofappropriate cells are plated in a 6-well tissue culture dish containing3 mL of complete, supplemented culture media and are grown in a 37° C.incubator under 5% carbon dioxide until the cells reach a densityappropriate for transduction. Cells are inoculated with approximately 4μL of the adenoviral stock (approximately 5×10⁸ pfu/ml) and areincubated for approximately 24 hours in a 37° C. incubator under 5%carbon dioxide. The tranduction media is replaced with 3 mL of freshcomplete, supplemented media and cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 24 hours. Thetransduced cells can be used to conduct a BoNT/C1 activity assay using aSyntaxin-GFP substrate. For greater details on procedures described inthis example, see ViraPower™ Adenoviral Expression System InstructionManual 25-0543 version A, Invitrogen, Inc., (Jul. 15, 2002).

8. Generation of Cells Containing a BoNT/C1 Substrate by LentiviralTransduction

8a. Construction of pLenti6Ubc/V5-Syntaxin-GFP

To make a pLenti6Ubc/V5-Syntaxin-GFP construct encoding a BoNT/C1Syntaxin-GFP substrate, a nucleic acid fragment encoding the amino acidregion comprising a BoNT/C1 Syntaxin-GFP substrate is amplified from,e.g., pQBI-25/Syntaxin-1-GFP DNA (see Example I, 3a) using a polymerasechain reaction method and subcloned into a pCR2.1 vector using the TOPO®TA cloning method (Invitrogen, Inc, Carlsbad, Calif.). The forward andreverse oligonucleotide primers used for this reaction are designed toinclude unique restriction enzyme sites useful for subsequent subcloningsteps. The resulting pCR2.1/Syntaxin-GFP construct is digested withrestriction enzymes that 1) excise the insert containing the entire openreading frame encoding the Syntaxin-GFP peptide; and 2) enable thisinsert to be operably-linked to a pLenti6Ubc/V5 vector (Invitrogen,Inc., Carlsbad, Calif.). This insert is subcloned using a T4 DNA ligaseprocedure into a pLenti6Ubc/V5 vector that is digested with appropriaterestriction endonucleases to yield pLenti6Ubc/V5-Syntaxin-GFP. Theligation mixture is transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shockmethod, plated on 1.5% Luria-Bertani agar plates (pH 7.0) containing 100μg/mL of Ampicillin, and placed in a 37° C. incubator for overnightgrowth. Bacteria containing expression constructs are identified asAmpicillin resistant colonies. Candidate constructs are isolated usingan alkaline lysis plasmid mini-preparation procedure and analyzed byrestriction endonuclease digest mapping to determine the presence andorientation of the inset. This cloning strategy yields a mammalianexpression construct encoding the Syntaxin-GFP operably-linked to theexpression elements of the pLenti6Ubc/V5 vector an amino-terminal V5peptide.

8b. Production of a Lentiviral Stock ContainingpLenti6Ubc/V5-Syntaxin-GFP

To produce a lentiviral stock containing an expression constructencoding a BoNT/C1 Syntaxin-GFP substrate, a 3.0 mL transfectionsolution is prepared by adding 1.5 mL of OPTI-MEM Reduced Serum Mediumcontaining 36 μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.)incubated at room temperature for 5 minutes to 1.5 mL of OPTI-MEMReduced Serum Medium containing 3 μg of an expression construct encodinga BoNT/C1 Syntaxin-GFP substrate, such as, e.g.,pLenti6Ubc/V5-Syntaxin-GFP and 9 μg of ViraPower™ Pacakaging Mix. Afteran approximately 20 minute incubation at room temperature, the DNA-lipidcomplexes are added to a 10 cm tissue culture plate containing 5 mLOPTI-MEM Reduced Serum Medium. A 5 mL cell suspension containingapproximately 6×10⁶ 293A cells are then added to DNA-lipid complex mediaand grown in a 37° C. incubator under 5% carbon dioxide overnight.Transfection media is replaced with 10 mL of complete Dulbecco'sModified Eagle Media (DMEM), supplemented with 10% fetal bovine serum(FBS), 2 mM glutamine (Invitrogen, Inc, Carlsbad, Calif.), 1 mM sodiumpyruvate (Invitrogen, Inc, Carlsbad, Calif.), 1× penicillin/streptomycinsolution (Invitrogen, Inc, Carlsbad, Calif.) and 1×MEM non-essentialamino acids solution (Invitrogen, Inc, Carlsbad, Calif.), and grown in a37° C. incubator under 5% carbon dioxide for approximately 24-48 hours.The lentiovirus-containing cells are harvested by detaching the cellsusing the culture media and scraping cells from the culture plate.Detached cells and media are transferred to a 15 mL tube and centrifuged(5,000×g at 20° C. for 15 minutes) to pellet the cellular debris. Theclarified supernatant containing the lentiviral particles is transferredto 2 mL cryovials in 1 mL aliquots and should contain approximately5×10⁵ to 2×10⁷ tu/mL of lentiviral particles. Aliquots can be stored at−80° C. until needed.

8c. Transduction of Cells with an Lentiviral Stock Containing apLenti6Ubc/V5-Syntaxin-GFP

To transduce cells with the lentiviral stock containing an expressionconstruct encoding a BoNT/C1 Syntaxin-GFP substrate, a suitable density(1.5×10⁵ to 1.5×10⁶) of appropriate cells is plated in a 6-well tissueculture dish containing 3 mL of complete, supplemented culture media andthe cells are grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach a density of about 5×10⁵ cells/ml (6-16 hours). Cellsare inoculated with the lentiviral stock containing an expressionconstruct encoding a BoNT/C1 Syntaxin-GFP substrate, such as, e.g.,pLenti6Ubc/V5-Syntaxin-GFP, using a suitable multiplicity of infectionand are incubated for approximately 16-24 hours in a 37° C. incubatorunder 5% carbon dioxide. The tranduction media is replaced with 3 mL offresh complete, supplemented media and cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 24-48 hours. Thetransduced cells can be used to conduct a BoNT/C1 activity assay using aSyntaxin-GFP substrate. For greater details on procedures described inthis example, see ViraPower™ Lentiviral Expression System InstructionManual 25-0501 version E, Invitrogen, Inc., (Dec. 8, 2003).

9. Generation of Cells Containing a BoNT/C1 Substrate by ProteinTransformation

9a. Expression of a Syntaxin-GFP Substrate Using a Bacterial Cell Line

To express a BoNT/C1 Syntaxin-GFP substrate using bacteria, anexpression construct encoding a BoNT/C1 Syntaxin-GFP substrate, such as,e.g., pQBI-67/Syntaxin-1-GFP as described above in Example I, 3b, isintroduced into chemically competent E. coli BL21 (DE3) cells(Invitrogen, Inc, Carlsbad, Calif.) using a heat-shock transformationprotocol. The heat-shock reaction is then plated onto 1.5% Luria-Bertaniagar plates (pH 7.0) containing 100 μg/mL of Ampicillin and placed in a37° C. incubator for overnight growth. A single Ampicillin-resistantcolony of transformed E. coli containing pQBI-67/Syntaxin-GFP is used toinoculate a 15 mL test tube containing 3.0 mL Luria-Bertani media, (pH7.0) containing 100 μg/mL of Ampicillin which is then placed in a 37° C.incubator, shaking at 250 rpm, for overnight growth. The resultingovernight starter culture is used to inoculate a 1.0 L baffled flaskcontaining 100 mL Luria-Bertani media, (pH 7.0) containing 100 μg/mL ofAmpicillin at a dilution of 1:1000. This culture is grown in a 32° C.incubator shaking at 250 rpm for approximately 6.5 hours until mid-logphase is reached (OD₆₀₀ of about 0.6-0.8). Protein expression is theninduced by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) andthe culture is placed in a 32° C. incubator shaking at 250 rpm forovernight expression. Cells are harvested by centrifugation (4,000 rpmat 4° C. for 20-30 minutes) to pellet the cells. The supernatant isdiscarded and the cell pellet is used immediately for subsequent steps,or the pellet is stored at −80° C. until needed.

9b. Expression of a Syntaxin-GFP Substrate Using a Mammalian Cell Line

To express a BoNT/C1 Syntaxin-GFP substrate using a mammalian cell line,a suitable density (1.0×10⁵ to 1.0×10⁶) of appropriate cells is platedin a 35 mm tissue culture dish containing 3 mL of complete, supplementedculture media and the cells are grown in a 37° C. incubator under 5%carbon dioxide until the cells reach a density of about 5×10⁵ cells/ml(6-16 hours). A 500 μL transfection solution is prepared by adding 250μL of OPTI-MEM Reduced Serum Medium containing 15 μL of LipofectAmine2000 (Invitrogen, Carlsbad, Calif.) incubated at room temperature for 5minutes to 250 μL of OPTI-MEM Reduced Serum Medium containing 5 μg of anexpression construct encoding a BoNT/C1 Syntaxin-GFP substrate, such as,e.g., pQBI-25/Syntaxin-1-GFP (see Example I, 3a). This transfection isincubated at room temperature for approximately 20 minutes. Thecomplete, supplemented culture media is replaced with 2 mL of OPTI-MEMReduced Serum Medium and the 500 μL transfection solution is added tothe cells and the cells are incubated in a 37° C. incubator under 5%carbon dioxide for approximately 16 hours. Transfection media isreplaced with 3 mL of fresh complete, supplemented culture media andcells are incubated in a 37° C. incubator under 5% carbon dioxide forapproximately 48 hours. Cells are harvest by rinsing cells once with 3.0mL of 100 mM phosphate-buffered saline, pH 7.4 and detaching rinsedcells by adding 500 μl of 100 mM phosphate-buffered saline, pH 7.4 andscraping cells from the culture plate. Detached cells are transferred toa 1.5 mL test tube and are pelleted by microcentrifugation (10,000×g at4° C. for 5 minutes). The supernatant is discarded and the cell pelletis used immediately for subsequent steps, or the pellet is stored at−80° C. until needed.

9c. Purification of a Syntaxin-GFP Substrate

To purify a Syntaxin-GFP substrate, a cell pellet expressing anexpression construct encoding a BoNT/C1 Syntaxin-GFP substrate, such as,e.g., either a pQBI-67/Syntaxin-1-GFP or a pQBI-25/Syntaxin-1-GFPconstruct, is resuspended in 10 mL of Tris-EDTA Buffer (10 mM Tris-HCl,pH 8.0; 1 mM EDTA, pH 8.0), containing 1 mg/mL of lysozyme and the cellsare lysed using three freeze-thaw rounds consisting of −80° C. for 5minutes then 37° C. for 5 minutes. The cell lysate is centrifuged(5,000×g at 4° C. for 15 minutes) to pellet the cellular debris and thesupernatant is transferred to a new tube containing an equal volume ofColumn Binding Buffer (4 M ammonium sulfate). A hydrophobic interactionchromatography (HIC) column is prepared using a 20 mL Econo-Pac columnsupport (Bio-Rad Laboratories, Hercules, Calif.) that is packed with2.5-5.0 mL of methyl HIC resin (Bio-Rad Laboratories, Hercules, Calif.),which is then equilibrated by rinsing with 5 column volumes of ColumnEquilibration Buffer (2 M ammonium sulfate). The clarified lysate isapplied slowly to the equilibrated column by gravity flow (approximately0.25-0.3 mL/minute). The column is then washed with 5 column volumes ofColumn Wash Buffer (1.3 M ammonium sulfate). The Syntaxin-GFP substrateis eluted with 20-30 mL of Column Elution Buffer (10 mM TE Buffer) andis collected in approximately twelve 1 mL fractions. The progress of theSyntaxin-GFP substrate sample through the column as well as whichelution fractions contain the sample is monitored using an ultravioletlight from a hand-held transilluminator. The amount of Syntaxin-GFPsubstrate contained in each elution fraction is determined by a Bradforddye assay. In this procedure, 20 μL aliquot from each 1.0 mL fraction iscombined with 200 μL of Bio-Rad Protein Reagent (Bio-Rad Laboratories,Hercules, Calif.), diluted 1 to 4 with deionized, distilled water, andthen the intensity of the colorimetric signal is measured using aspectrophotometer. The five fractions with the strongest signal areconsidered to comprise the elution peak and are pooled. Total proteinyield are determined by estimating the total protein concentration ofthe pooled peak elution fractions using bovine gamma globulin as astandard (Bio-Rad Laboratories, Hercules, Calif.). The amount ofSyntaxin-GFP substrate is adjusted to a protein concentration ofapproximately 100 ng/mL.

9d. Protein Transformation of a Syntaxin-GFP Substrate

To transform a Syntaxin-GFP substrate into a mammalian cell line, asuitable density (1.0×10⁵ to 1.0×10⁶) of appropriate cells is plated ina 35 mm tissue culture dish containing 3 mL of complete, supplementedculture media and the cells are grown in a 37° C. incubator under 5%carbon dioxide until the cells reach a density of about 5×10⁵ cells/ml(6-16 hours). A 200 μL protein transfection solution is prepared byadding 100 μL of distilled water containing 6 μL of Chariot™ proteindelivery agent (Active Motif, Carlsbad, Calif.) to 100 μL of 100 mMphosphate-buffered saline, pH 7.4 containing 1 μg of a Syntaxin-GFPsubstrate and this solution is incubated at room temperature forapproximately 30 minutes. After incubation, the cells are washed once byrinsing cells with 3.0 mL of 100 mM phosphate-buffered saline, pH 7.4.The 200 μL protein transfection solution is added to the washed cells,followed by 400 μL of OPTI-MEM Reduced Serum Medium and the cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 1 hour. Add 1 mL of fresh complete, supplemented culturemedia to the cells and incubate in a 37° C. incubator under 5% carbondioxide. After 1-2 hours, the transformed cells can be used to conduct aBoNT/C1 activity assay using a Syntaxin-GFP substrate.

Example V Generation of Cells Stably Containing a Clostridial ToxinSubstrate

1. Generation of Cells Stably Containing a BoNT/A, BoNT/C1 or BoNT/ESubstrate

1a. Stably Transformed Neuro-2A Cells Using a Recombinant Crossing-OverProcedure

To generate a stably-integrated cell line expressing a BoNT/A, BoNT/C1or BoNT/E SNAP-25 substrate using a crossing over procedure,approximately 1.5×10⁵ Neuro-2A cells were plated in a 35 mm tissueculture dish containing 3 mL of complete EMEM, supplemented with 10%FBS, 2 mM glutamine

(Invitrogen, Inc, Carlsbad, Calif.), 1 mM sodium pyruvate (Invitrogen,Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate and 1×MEMnon-essential amino acids solution (Invitrogen, Inc, Carlsbad, Calif.),and grown in a 37° C. incubator under 5% carbon dioxide until the cellsreached a density of about 5×10⁵ cells/ml. A 500 μL transfectionsolution was prepared by adding 250 μL of OPTI-MEM Reduced Serum Mediumcontaining 15 μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.)incubated at room temperature for 5 minutes to 250 μL of OPTI-MEMReduced Serum Medium containing 5 μg of an expression construct encodinga BoNT/A, BoNT/C1 or BoNT/E, substrate, such as, e.g.,pQBI-25/SNAP25₂₀₆-GFP (see EXAMPLE I, 1). This transfection wasincubated at room temperature for approximately 20 minutes. Thecomplete, supplemented EMEM media was replaced with 2 mL of OPTI-MEMReduced Serum Medium and the 500 μL transfection solution was added tothe Neuro-2A cells and the cells incubated in a 37° C. incubator under5% carbon dioxide for approximately 16 hours. Transfection media wasreplaced with 3 mL of fresh complete, supplemented EMEM and cells wereincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 48 hours. Media was replaced with 3 mL of fresh completeEMEM, containing approximately 5 μg/mL of G418, 10% FBS, 2 mM glutamine(Invitrogen, Inc, Carlsbad, Calif.), 1 mM sodium pyruvate (Invitrogen,Inc, Carlsbad, Calif.). 1.5 g/L sodium bicarbonate and 1×MEMnon-essential amino acids solution (Invitrogen, Inc, Carlsbad, Calif.).Cells were incubated in a 37° C. incubator under 5% carbon dioxide forapproximately 4 weeks, with old media being replaced with freshG418-selective, complete, supplemented EMEM every 4 to 5 days. OnceG418-resistant Neuro-2A colonies were established, resistant clones werereplated to new 35 mm culture plates containing fresh G418-selective,complete, supplemented EMEM, until these cells reached a density of 6 to20×10⁵ cells/mL.

To test for expression of SNAP-25₂₀₆-GFP from isolated Neuro-2A celllines that stably-integrated expression construct encoding a BoNT/A,BoNT/C1 or BoNT/E, substrate, such as, e.g., pQBI-25/SNAP25₂₀₆-GFP (seeExample I, 1a), approximately 1.5×10⁵ Neuro-2A cells from each cell linewere plated in a 35 mm tissue culture dish containing 3 mL ofG418-selective, complete, supplemented EMEM and grown in a 37° C.incubator under 5% carbon dioxide until cells reached a density of about5×10⁵ cells/ml (6-16 hours). Media was replaced with 3 mL of freshG418-selective, complete, supplemented EMEM and cells were incubated ina 37° C. incubator under 5% carbon dioxide. After 48 hours, the cellswere harvested by rinsing the cells once with 3.0 mL of 100 mMphosphate-buffered saline, pH 7.4 and lysed with a buffer containing62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate (SDS). Lysed cells werecentrifuged at 5000 rpm for 10 min at 4° C. to eliminate debris and thesupernatants were transferred to fresh siliconized tubes. Proteinconcentrations were measured by Bradford's method and resuspended in1×SDS sample buffer at 1 mg/ml or higher concentration.

To detect for the presence of the SNAP-25-GFP substrate, samples wereboiled for 5 min, and 40 μl aliquots were separated by MOPSpolyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Trisprecast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Separated peptides were transferredfrom the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen,Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SDsemi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories,Hercules, Calif.). PVDF membranes were blocked by incubating at roomtemperature for 2 hours in a solution containing 25 mM Tris-BufferedSaline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride),0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovineserum albumin, 5% nonfat dry milk. Blocked membranes were incubated at4° C. for overnight in Tris-Buffered Saline TWEEN-20® (25 mMTris-Buffered Saline, 0.1% TWEEN-20′, polyoxyethylene (20) sorbitanmonolaureate) containing a 1:50,000 dilution of mouse monoclonalanti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals, Lutherville,Md.). Primary antibody probed blots were washed three times for 15minutes each time in Tris-Buffered Saline TWEEN-20®. Washed membraneswere incubated at room temperature for 2 hours in Tris-Buffered SalineTWEEN-20® containing a 1:20,000 dilution of goat polyclonal anti-mouseimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) as a secondary antibody. Secondary antibody-probed blots werewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled SNAP-25₂₀₆-GFP substrate wasvisualized using the ECL Plus™ Western Blot Detection System (AmershamBiosciences, Piscataway, N.J.) and the membrane was imaged and substratequantitated with a Typhoon 9410 Variable Mode Imager and Imager Analysissoftware (Amersham Biosciences, Piscataway, N.J.). The choice of pixelsize (100 to 200 pixels) and PMT voltage settings (350 to 600, normally400) depended on the individual blot. X isolated Neuro-2A cell lineswere identified that stably integrated and expressed the SNAP-25₂₀₆-GFPsubstrate of SEQ ID NO: 1.

To determine the subcellular localization of the SNAP-25-GFP substratefrom isolated Neuro-2A cell lines that stably-integrated an expressionconstruct encoding a BoNT/A, BoNT/C1 or BoNT/E, substrate, such as,e.g., pQBI-25/SNAP25₂₀₆-GFP (see Example I, 1a), approximately 1.5×10⁵Neuro-2A cells from each cell line were plated in a 35 mm tissue culturedish containing 3 mL of G418-selective, complete, supplemented EMEM andgrown in a 37° C. incubator under 5% carbon dioxide until cells reacheda density of about 5×10⁵ cells/ml (6-16 hours). Media was replaced with3 mL of fresh G418-selective, complete, supplemented EMEM and cells wereincubated in a 37° C. incubator under 5% carbon dioxide. After 24-48hours, living cells were observation using a fluorescence invertedmicroscope. X isolated Neuro-2A cell lines were detected to have thelocalization of the GFP fluorescence in the cell membrane indicatingthat the expressed SNAP25₂₀₆-GFP in these isolated Neuro-2A cell lineswas correctly targeted to the cell membrane. Stably transduced cells canbe used to conduct a BoNT/A, BoNT/C1 or BoNT/E activity assay.

1b. Stably Transduced SH-SY5Y Cells Using a Lentiviral Procedure

To generate a stably-integrated cell line expressing a BoNT/A, BoNT/C1or BoNT/E SNAP-25 substrate using a lentioviral procedure, about 1.5×10⁵SH-SY5Y cells are plated in a 6-well tissue culture dish containing 3 mLof complete 1:1 EMEM and Ham's F12 Media (EMEM:F12), supplemented with10% fetal bovine serum (FBS), 4 mM glutamine (Invitrogen, Inc, Carlsbad,Calif.), 1% sodium pyruvate (Invitrogen, Inc, Carlsbad, Calif.), 1.5 g/Lsodium bicarbonate, 1× penicillin/streptomycin solution (Invitrogen,Inc, Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37° C. incubatorunder 5% carbon dioxide until the cells reach a density of about 5×10⁵cells/ml (6-16 hours). Cells are inoculated with the lentioviral stockcontaining an expression construct encoding a BoNT/A, BoNT/C1 or BoNT/E,substrate, such as, e.g., pLenti6Ubc/V5-SNAP-25₂₀₆-GFP, as describedabove in Example IV, 2b, using a suitable multiplicity of infection andare incubated for approximately 16-24 hours in a 37° C. incubator under5% carbon dioxide. The tranduction media is replaced with 3 mL of freshcomplete, supplemented EMEM:F12 containing an appropriate amount ofBlasticidin. Cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 2 weeks, with old media being replaced withfresh Blasticidin-selective, complete, supplemented EMEM:F12 every 3 to4 days. Once Blasticidin-resistant SH-SY5Y colonies are established,resistant clones are replated to new 35 mm culture plates containingfresh Blasticidin-selective, complete, supplemented EMEM:F12, untilthese cells reached a density of 6 to 20×10⁵ cells/mL. For greaterdetails on procedures described in this example, see ViraPower™Lentiviral Expression System Instruction Manual 25-0501 version E,Invitrogen, Inc., (Dec. 8, 2003).

The presence of the SNAP-25-GFP substrate in isolated cell lines will bedetermined by Western blot analysis as describes above in Example V, 1a.The subcellular localization of the SNAP-25₂₀₆-GFP substrate in isolatedcell lines will be determined by fluorescence microscopy as describesabove in Example V, 1a. Stably transduced cells can be used to conduct aBoNT/A, BoNT/C1 or BoNT/E activity assay.

1c. Stably Transduced SK-N-DZ Cells Using a Recombinant Crossing-OverProcedure

To generate a stably-integrated cell line expressing a BoNT/A, BoNT/C1or BoNT/E SNAP-25 substrate using a crossing over procedure,approximately 1.5×10⁵ SK-N-DZ cells were plated in a 35 mm tissueculture dish containing 3 mL of complete DMEM, supplemented with 10%FBS, 4 mM glutamine

(Invitrogen, Inc, Carlsbad, Calif.) and 1×MEM non-essential amino acidssolution (Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37° C.incubator under 5% carbon dioxide until the cells reached a density ofabout 5×10⁵ cells/ml. A 500 μL transfection solution was prepared byadding 250 μL of OPTI-MEM Reduced Serum Medium containing 15 μL ofLipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated at roomtemperature for 5 minutes to 250 μL of OPTI-MEM Reduced Serum Mediumcontaining 5 μg of an expression construct encoding a BoNT/A, BoNT/C1 orBoNT/E, substrate, such as, e.g., pQBI-25/SNAP25₂₀₆-GFP (see Example I,1a). This transfection was incubated at room temperature forapproximately 20 minutes. The complete, supplemented DMEM media wasreplaced with 2 mL of OPTI-MEM Reduced Serum Medium and the 500 μLtransfection solution was added to the SK-N-DZ cells and the cellsincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 16 hours. Transfection media was replaced with 3 mL offresh complete, supplemented DMEM and cells were incubated in a 37° C.incubator under 5% carbon dioxide for approximately 48 hours. Media wasreplaced with 3 mL of fresh complete DMEM, containing approximately 5μg/mL of G418, 10% FBS, 1× penicillin/streptomycin solution (Invitrogen,Inc, Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.). Cells were incubated in a 37° C.incubator under 5% carbon dioxide for approximately 4 weeks, with oldmedia being replaced with fresh G418 selective, complete, supplementedDMEM every 4 to 5 days. Once G418-resistant SK-N-DZ colonies wereestablished, resistant clones were replated to new 35 mm culture platescontaining fresh complete DMEM, supplemented with approximately 5 μg/mLof G418, 10% FBS, 1× penicillin/streptomycin solution (Invitrogen, Inc,Carlsbad, Calif.) and 1×MEM non-essential amino acids solution(Invitrogen, Inc, Carlsbad, Calif.), until these cells reached a densityof 6 to 20×10⁵ cells/mL.

To test for expression of SNAP-25-GFP from isolated SK-N-DZ cell linesthat stably-integrated an expression construct encoding a BoNT/A,BoNT/C1 or BoNT/E, substrate, such as, e.g., pQBI-25/SNAP25₂₀₆-GFP (seeExample I, 1a), approximately 1.5×10⁵ SK-N-DZ cells from each cell linewere plated in a 35 mm tissue culture dish containing 3 mL ofG418-selective, complete, supplemented DMEM and grown in a 37° C.incubator under 5% carbon dioxide until cells reached a density of about5×10⁵ cells/ml (6-16 hours). Media was replaced with 3 mL of freshG418-selective, complete, supplemented DMEM and cells were incubated ina 37° C. incubator under 5% carbon dioxide. After 48 hours, the cellswere harvested by rinsing the cells once with 3.0 mL of 100 mMphosphate-buffered saline, pH 7.4 and lysed with a buffer containing62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate (SDS). Lysed cells werecentrifuged at 5000 rpm for 10 min at 4° C. to eliminate debris and thesupernatants were transferred to fresh siliconized tubes. Proteinconcentrations were measured by Bradford's method and resuspended in1×SDS sample buffer at 1 mg/ml or higher concentration.

To detect for the presence of the SNAP-25₂₀₆-GFP substrate, samples wereboiled for 5 min, and 40 μl aliquots were separated by MOPSpolyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Trisprecast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Separated peptides were transferredfrom the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen,Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SDsemi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories,Hercules, Calif.). PVDF membranes were blocked by incubating at roomtemperature for 2 hours in a solution containing 25 mM Tris-BufferedSaline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride),0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovineserum albumin, 5% nonfat dry milk. Blocked membranes were incubated at4° C. for overnight in Tris-Buffered Saline TWEEN-20° (25 mMTris-Buffered Saline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitanmonolaureate) containing a 1:50,000 dilution of mouse monoclonalanti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals, Lutherville,Md.). Primary antibody probed blots were washed three times for 15minutes each time in Tris-Buffered Saline TWEEN-20®. Washed membraneswere incubated at room temperature for 2 hours in Tris-Buffered SalineTWEEN-20® containing a 1:20,000 dilution of goat polyclonal anti-mouseimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) as a secondary antibody. Secondary antibody-probed blots werewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled SNAP-25₂₀₆-GFP substrate wasvisualized using the ECL Plus™ Western Blot Detection System (AmershamBiosciences, Piscataway, N.J.) and the membrane was imaged and substratequantitated with a Typhoon 9410 Variable Mode Imager and Imager Analysissoftware (Amersham Biosciences, Piscataway, N.J.). The choice of pixelsize (100 to 200 pixels) and PMT voltage settings (350 to 600, normally400) depended on the individual blot. X isolated SK-N-DZ cell lines wereidentified that stably integrated and expressed the SNAP-25₂₀₆-GFPsubstrate of SEQ ID NO: 1.

To determine the subcellular localization of the SNAP-25₂₀₆-GFPsubstrate from isolated SK-N-DZ cell lines that stably-integrated anexpression construct encoding a BoNT/A, BoNT/C1 or BoNT/E, substrate,such as, e.g., pQBI-25/SNAP25₂₀₆-GFP (see Example I, 1a), approximately1.5×10⁵ SK-N-DZ cells from each cell line were plated in a 35 mm tissueculture dish containing 3 mL of G418-selective, complete, supplementedDMEM and grown in a 37° C. incubator under 5% carbon dioxide until cellsreached a density of about 5×10⁵ cells/ml (6-16 hours). Media wasreplaced with 3 mL of fresh G418-selective, complete, supplemented DMEMand cells were incubated in a 37° C. incubator under 5% carbon dioxide.After 24-48 hours, living cells were observation using a fluorescenceinverted microscope. X isolated SK-N-DZ cell lines were detected to havethe localization of the GFP fluorescence in the cell membrane indicatingthat the expressed SNAP25₂₀₆-GFP in these isolated SK-N-DZ cell lineswas correctly targeted to the cell membrane. Stably transduced cells canbe used to conduct a BoNT/A, BoNT/C1 or BoNT/E activity assay using aSNAP-25-GFP substrate.

1d. Stably Transduced SK-N-DZ Cells Using a Lentiviral Procedure

To generate a stably-integrated cell line expressing a BoNT/A, BoNT/C1or BoNT/E SNAP-25 substrate using a lentioviral procedure, approximately1.5×10⁵ SK-N-DZ cells are plated in a 6-well tissue culture dishcontaining 3 mL of complete DMEM, supplemented with 10% FBS, 4 mMglutamine (Invitrogen, Inc, Carlsbad, Calif.) and 1×MEM non-essentialamino acids solution (Invitrogen, Inc, Carlsbad, Calif.), and grown in a37° C. incubator under 5% carbon dioxide until the cells reach a densityof about 5×10⁵ cells/ml (6-16 hours). Cells are inoculated with thelentiviral stock containing an expression construct encoding a BoNT/A,BoNT/C1 or BoNT/E substrate, such as, e.g.,pLenti6Ubc/V5-SNAP-25₂₀₆-GFP, as described above in Example IV, 2b,using a suitable multiplicity of infection and are incubated forapproximately 16-24 hours in a 37° C. incubator under 5% carbon dioxide.The tranduction media is replaced with 3 mL of fresh complete,supplemented EMEM:F12 containing an appropriate amount of Blasticidin.Cells are incubated in a 37° C. incubator under 5% carbon dioxide forapproximately 2 weeks, with old media being replaced with freshBlasticidin-selective, complete, supplemented EMEM:F12 every 3 to 4days. Once Blasticidin-resistant SH-SY5Y colonies are established,resistant clones are replated to new 35 mm culture plates containingfresh Blasticidin-selective, complete, supplemented EMEM:F12, untilthese cells reached a density of 6 to 20×10⁵ cells/mL. For greaterdetails on procedures described in this example, see ViraPower™Lentiviral Expression System Instruction Manual 25-0501 version E,Invitrogen, Inc., (Dec. 8, 2003).

The presence of the SNAP-25₂₀₆-GFP substrate in isolated cell lines willbe determined by Western blot analysis as describes above in Example V,1a. The subcellular localization of the SNAP-25₂₀₆-GFP substrate inisolated cell lines will be determined by fluorescence microscopy asdescribes above in Example V, 1a. Stably transduced cells can be used toconduct a BoNT/A, BoNT/C1 or BoNT/E activity assay using a SNAP-25-GFPsubstrate.

2. Generation of Cells Stably Containing a BoNT/B, BoNT/D, BoNT/F,BoNT/G or TeNT Substrate

2a. Stably Transformed Cells Using a Recombinant Crossing-Over Procedure

To generate a stably-integrated cell line expressing a BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT VAMP substrate using a crossing over procedure, asuitable density (1×10⁵ to 1×106⁶ cells) of appropriate cells are platedin a 35 mm tissue culture dish containing 3 mL of complete, supplementedculture media and grown in a 37° C. incubator under 5% carbon dioxideuntil the cells reached a density appropriate for transfection. A 500 μLtransfection solution is prepared by adding 250 μL of OPTI-MEM ReducedSerum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 5 μg of expression constructencoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP substrate, suchas, e.g., pQBI-25/VAMP-1-GFP, pQBI-25/VAMP-2-GFP or pQBI-25/VAMP-3-GFP(see Examples I, 2a; I, 2b; or I, 2c). This transfection was incubatedat room temperature for approximately 20 minutes. The complete,supplemented media is replaced with 2 mL of OPTI-MEM Reduced SerumMedium and the 500 μL transfection solution is added to the cells andthe cells are incubated in a 37° C. incubator under 5% carbon dioxidefor approximately 16 hours. Transfection media is replaced with 3 mL offresh complete, supplemented culture media and the cells are incubatedin a 37° C. incubator under 5% carbon dioxide for approximately 48hours. Media is replaced with 3 mL of fresh complete, supplementedculture media, containing approximately 5 μg/mL of G418. Cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 4 weeks, with old media being replaced with fresh G418selective, complete, supplemented media every 4 to 5 days. OnceG418-resistant colonies are established, resistant clones are replatedto new 35 mm culture plates containing fresh complete culture media,supplemented with approximately 5 μg/mL of G418 until these cellsreached a density of 6 to 20×10⁵ cells/mL.

To test for expression of a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNTVAMP-GFP from isolated cell lines that stably-integrated an expressionconstruct encoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFPsubstrate, such as, e.g., pQBI-25/VAMP-1-GFP, pQBI-25/VAMP-2-GFP orpQBI-25/VAMP-3-GFP (see Examples I, 2a; I, 2b; or I, 2c), approximately1.5×10⁵ cells from each cell line are plated in a 35 mm tissue culturedish containing 3 mL of G418-selective, complete, supplemented DMEM andare grown in a 37° C. incubator under 5% carbon dioxide until cellsreached a density of about 5×10⁵ cells/ml (6-16 hours). Media isreplaced with 3 mL of fresh G418-selective, complete, supplementedculture media and cells are incubated in a 37° C. incubator under 5%carbon dioxide. After 48 hours, the cells are harvested by rinsing thecells once with 3.0 mL of 100 mM phosphate-buffered saline, pH 7.4 andare lysed with a buffer containing 62.6 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl), pH6.8 and 2% sodium lauryl sulfate (SDS). Lysed cells are centrifuged at5000 rpm for 10 min at 4° C. to eliminate debris and the supernatantsare transferred to fresh siliconized tubes. Protein concentrations aremeasured by Bradford's method and are resuspended in 1×SDS sample bufferat 1 mg/ml or higher concentration.

To detect for the presence of the VAMP-GFP substrate, samples areseparated by MOPS polyacrylamide gel electrophoresis and analyzed byWestern blotting procedures as described above in Examples II, 2a; II,4a; II, 6a; II, 7a; and II, 8a, in order to identify cell lines thathave stably integrated and express the VAMP-GFP substrate.

To determine the subcellular localization of the VAMP-GFP substrate fromisolated cell lines that stably-integrated an expression constructencoding a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT VAMP-GFP substrate,such as, e.g., pQBI-25/VAMP-1-GFP, pQBI-25/VAMP-2-GFP orpQBI-25/VAMP-3-GFP (see Examples I, 2a; I, 2b; or I, 2c), approximately1.5×10⁵ cells from each cell line are plated in a 35 mm tissue culturedish containing 3 mL of G418-selective, complete, supplemented culturemedia and are grown in a 37° C. incubator under 5% carbon dioxide untilcells reached a density of about 5×10⁵ cells/ml (6-16 hours). Media isreplaced with 3 mL of fresh G418-selective, complete, supplementedculture media and cells are incubated in a 37° C. incubator under 5%carbon dioxide. After 24-48 hours, living cells are observation using afluorescence inverted microscope in order to identify isolated celllines that exhibit GFP fluorescence localized to the cell membrane,thereby indicating that the expressed VAMP-GFP in these isolated celllines is correctly targeted to the cell membrane. Stably transducedcells can be used to conduct a BoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNTactivity assay using a VAMP-GFP substrate.

2b. Stably Transduced Cells Using a Lentiviral Procedure

To generate a stably-integrated cell line expressing a BoNT/B, BoNT/D,BoNT/F, BoNT/G or TeNT VAMP substrate using a lentiviral procedure, asuitable density (1×10⁵ to 1×106⁶ cells) of appropriate cells are platedin a 6-well tissue culture dish containing 3 mL of complete,supplemented culture media and are grown in a 37° C. incubator under 5%carbon dioxide until the cells reach a density appropriate fortransduction. Cells are inoculated with the lentiviral stock, asdescribed above in Example IV, 5b, using a suitable multiplicity ofinfection and are incubated for approximately 16-24 hours in a 37° C.incubator under 5% carbon dioxide. The tranduction media is replacedwith 3 mL of fresh complete, supplemented media containing anappropriate amount of Blasticidin. Cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 2 weeks, with oldmedia being replaced with fresh Blasticidin-selective, complete,supplemented media every 3 to 4 days. Once Blasticidin-resistantcolonies are established, resistant clones are replated to new 35 mmculture plates containing fresh Blasticidin-selective, complete,supplemented media, until these cells reached a density of 6 to 20×10⁵cells/mL. For greater details on procedures described in this example,see ViraPower™ Lentiviral Expression System Instruction Manual 25-0501version E, Invitrogen, Inc., (Dec. 8, 2003).

The presence of the VAMP-GFP substrate in isolated cell lines will bedetermined by Western blot analysis as describes above in Example V, 2a.The subcellular localization of the VAMP-GFP substrate in isolated celllines will be determined by fluorescence microscopy as describes abovein Example V, 2a. Stably transduced cells can be used to conduct aBoNT/B, BoNT/D, BoNT/F, BoNT/G or TeNT activity assay using a VAMP-GFPsubstrate.

3. Generation of Cells Stably Containing a BoNT/C1 Syntaxin Substrate

3a. Stably Transformed Cells Using a Recombinant Crossing-Over Procedure

To generate a stably-integrated cell line expressing a BoNT/C1 Syntaxinsubstrate using a crossing over procedure, a suitable density (1×10⁵ to1×106⁶ cells) of appropriate cells are plated in a 35 mm tissue culturedish containing 3 mL of complete, supplemented culture media and grownin a 37° C. incubator under 5% carbon dioxide until the cells reached adensity appropriate for transfection. A 500 μL transfection solution isprepared by adding 250 μL of OPTI-MEM Reduced Serum Medium containing 15μL of LipofectAmine 2000 (Invitrogen, Carlsbad, Calif.) incubated atroom temperature for 5 minutes to 250 μL of OPTI-MEM Reduced SerumMedium containing 5 μg of expression construct encoding a BoNT/C1Syntaxin substrate, such as, e.g., pQBI-25/Syntaxin-1-GFP (see ExampleI, 3a). This transfection was incubated at room temperature forapproximately 20 minutes. The complete, supplemented media is replacedwith 2 mL of OPTI-MEM Reduced Serum Medium and the 500 μL transfectionsolution is added to the cells and the cells are incubated in a 37° C.incubator under 5% carbon dioxide for approximately 16 hours.Transfection media is replaced with 3 mL of fresh complete, supplementedculture media and the cells are incubated in a 37° C. incubator under 5%carbon dioxide for approximately 48 hours. Media is replaced with 3 mLof fresh complete, supplemented culture media, containing approximately5 μg/mL of G418. Cells are incubated in a 37° C. incubator under 5%carbon dioxide for approximately 4 weeks, with old media being replacedwith fresh G418 selective, complete, supplemented media every 4 to 5days. Once G418-resistant colonies are established, resistant clones arereplated to new 35 mm culture plates containing fresh complete culturemedia, supplemented with approximately 5 μg/mL of G418 until these cellsreached a density of 6 to 20×10⁵ cells/mL.

To test for expression of a BoNT/C1 Synataxin-GFP from isolated celllines that stably-integrated an expression construct encoding a BoNT/C1Syntaxin-GFP substrate, such as, e.g., pQBI-25/Syntaxin-1-GFP (seeExample I, 3a), approximately 1.5×10⁵ cells from each cell line areplated in a 35 mm tissue culture dish containing 3 mL of G418-selective,complete, supplemented culture media and are grown in a 37° C. incubatorunder 5% carbon dioxide until cells reached a density of about 5×10⁵cells/ml (6-16 hours). Media is replaced with 3 mL of freshG418-selective, complete, supplemented culture media and cells areincubated in a 37° C. incubator under 5% carbon dioxide. After 48 hours,the cells are harvested by rinsing the cells once with 3.0 mL of 100 mMphosphate-buffered saline, pH 7.4 and are lysed with a buffer containing62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate (SDS). Lysed cells arecentrifuged at 5000 rpm for 10 min at 4° C. to eliminate debris and thesupernatants are transferred to fresh siliconized tubes. Proteinconcentrations are measured by Bradford's method and are resuspended in1×SDS sample buffer at 1 mg/ml or higher concentration.

To detect for the presence of the Syntaxin-GFP substrate, samples areseparated by MOPS polyacrylamide gel electrophoresis and analyzed byWestern blotting procedures as described above in Examples II, 3a, inorder to identify cell lines that have stably integrated and express theSyntaxin-GFP substrate.

To determine the subcellular localization of the Syntaxin-GFP substratefrom isolated cell lines that stably-integrated an expression constructencoding a BoNT/C1 Syntaxin-GFP substrate, such as, e.g.,pQBI-25/Syntaxin-1-GFP (see Example I, 3a), approximately 1.5×10⁵ cellsfrom each cell line are plated in a 35 mm tissue culture dish containing3 mL of G418-selective, complete, supplemented culture media and aregrown in a 37° C. incubator under 5% carbon dioxide until cells reacheda density of about 5×10⁵ cells/ml (6-16 hours). Media is replaced with 3mL of fresh G418-selective, complete, supplemented culture media andcells are incubated in a 37° C. incubator under 5% carbon dioxide. After24-48 hours, living cells are observation using a fluorescence invertedmicroscope in order to identify isolated cell lines that exhibit GFPfluorescence localized to the cell membrane, thereby indicating that theexpressed Syntaxin-GFP in these isolated cell lines is correctlytargeted to the cell membrane. Stably transduced cells can be used toconduct a BoNT/C1 activity assay using a Syntaxin-GFP substrate.

3b. Stably Transduced Cells Using a Lentiviral Procedure

To generate a stably-integrated cell line expressing a BoNT/C1 Syntaxinsubstrate using a lentiviral procedure, a suitable density (1×10⁵ to1×106⁶ cells) of appropriate cells are plated in a 6-well tissue culturedish containing 3 mL of complete, supplemented culture media and aregrown in a 37° C. incubator under 5% carbon dioxide until the cellsreach a density appropriate for transduction. Cells are inoculated withthe lentiviral stock, as described above in Example IV, 8b, using asuitable multiplicity of infection and are incubated for approximately16-24 hours in a 37° C. incubator under 5% carbon dioxide. Thetranduction media is replaced with 3 mL of fresh complete, supplementedmedia containing an appropriate amount of Blasticidin. Cells areincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 2 weeks, with old media being replaced with freshBlasticidin-selective, complete, supplemented media every 3 to 4 days.Once Blasticidin-resistant colonies are established, resistant clonesare replated to new 35 mm culture plates containing freshBlasticidin-selective, complete, supplemented media, until these cellsreached a density of 6 to 20×10⁵ cells/mL. For greater details onprocedures described in this example, see ViraPower™ LentiviralExpression System Instruction Manual 25-0501 version E, Invitrogen,Inc., (Dec. 8, 2003).

The presence of the Syntaxin-GFP substrate in isolated cell lines willbe determined by Western blot analysis as describes above in Example V,3a. The subcellular localization of the Syntaxin-GFP substrate inisolated cell lines will be determined by fluorescence microscopy asdescribes above in Example V, 3a. Stably transduced cells can be used toconduct a BoNT/C1 activity assay using a Syntaxin-GFP substrate.

Example VI Clostridial Toxin Activity Assays

1. BoNT/A Activity Assays

1a. Assay of BoNT/A Activity in a BOTOX® Product

To conduct a BoNT/A activity assay using a formulated botulinumneurotoxin product such as, e.g., a BOTOX® product, differentiatedNeuro-2A cells expressing a BoNT/A SNAP25₂₀₆-GFP substrate will beprepared, for example, using one of the methods as described above inExamples IV, 1 and V, 1. A standard curve will be obtained by treatingNeuro-2A cells with 0.001 nM, 0.002 nM, 0.005 nM, 0.01 nM, 0.02 nM or0.05 nM of Pure A (BTX-540; Metabiologics, Inc., Madison, Wis.), witheach of the concentrations run in triplicates in a 24 well plate.Simultaneously, separate wells in the same plate will be treated withBOTOX® dissolved in 1 ml of complete EMEM media to a final concentrationof approximately 0.0055 nM. Cells in three replicate wells will betreated with the contents of each resuspended BOTOX® vial. After sixhours, cells will be analyzed using the Typhoon 9140 Imager withexcitation at 484 nm and emission at 510 nm. The emissions at eachconcentration of Pure A and each BOTOX® sample will be calculated as apercentage of the untreated control (fluorescence measured at 510 nm ofnon-toxin treated cells). The experimentally-derived concentration ofBOTOX® for each vial will be extrapolated from the Pure A concentrationcurve using the value calculated from an average of three replicatewells.

1b. Assay of BoNT/A Activity in a Food Sample

To conduct a BoNT/A activity assay using a food sample such as, e.g., aprocessed food sample, differentiated Neuro-2A cells expressing a BoNT/ASNAP25₂₀₆-GFP substrate will be prepared, for example, using one of themethods as described above in Examples IV, 1 and V, 1. A standard curvewill be obtained by treating Neuro-2A cells with 0.001, 0.002, 0.005,0.01, 0.02 or 0.05 nM of Pure A (BTX-540; Metabiologics, Inc., Madison,Wis.), with each of the concentrations run in triplicates in a 24 wellplate. Simultaneously, separate wells in the same plate will be treatedwith a processed food sample from vials of BOTOX® diluted in 1 ml ofcomplete EMEM media. Cells in three replicate wells will be treated withthe contents of each diluted sample. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of Pure A andeach processed food sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally derived concentration of BoNT/A present inthe processed food sample will be extrapolated from the Pure Aconcentration curve using the value calculated from an average of threereplicate wells.

2. BoNT/B Activity Assays

2a. Assay of BoNT/B Activity in a Formulated BoNT/B Product

To conduct a BoNT/B activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/B product, cellsexpressing a BoNT/B VAMP-GFP substrate will be prepared, for example,using one of the methods as described above in Examples IV, 2 and V, 2.A standard curve will be obtained by treating cells with 0.001 nM, 0.002nM, 0.005 nM, 0.01 nM, 0.02 nM or 0.05 nM of BoNT/B (Metabiologics,Inc., Madison, Wis.), with each of the concentrations run in triplicatesin a 24 well plate. Simultaneously, separate wells in the same platewill be treated with formulated BoNT/B dissolved in 1 ml of completeculture media to a final concentration of approximately 0.0055 nM. Cellsin three replicate wells will be treated with the contents of eachresuspended formulated BoNT/B vial. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of BoNT/B andeach formulated BoNT/B sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally-derived concentration of formulated BoNT/Bfor each vial will be extrapolated from the BoNT/B concentration curveusing the value calculated from an average of three replicate wells.

2b. Assay of BoNT/B Activity in a Food Sample

To conduct a BoNT/B activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a BoNT/B VAMP-GFP substrate willbe prepared, for example, using one of the methods as described above inExamples IV, 2 and V, 2. A standard curve will be obtained by treatingcells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM of BoNT/B(Metabiologics, Inc., Madison, Wis.), with each of the concentrationsrun in triplicates in a 24 well plate. Simultaneously, separate wells inthe same plate will be treated with a processed food sample from vialsof formulated BoNT/B diluted in 1 ml of complete culture media. Cells inthree replicate wells will be treated with the contents of each dilutedsample. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/B and each processed food sample will becalculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). The experimentallyderived concentration of BoNT/B present in the processed food samplewill be extrapolated from the BoNT/B concentration curve using the valuecalculated from an average of three replicate wells.

3. BoNT/C1 Activity Assays

3a. Assay of BoNT/C1 Activity in a Formulated BoNT/C1 Product

To conduct a BoNT/C1 activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/C1 product, cellsexpressing a BoNT/C1 SNAP-25₂₀₆-GFP substrate or a BoNT/C1 Syntaxin-GFPsubstrate will be prepared, for example, using one of the methods asdescribed above in Examples IV, 3 and V, 3. A standard curve will beobtained by treating cells with 0.001 nM, 0.002 nM, 0.005 nM, 0.01 nM,0.02 nM or 0.05 nM of BoNT/B (Metabiologics, Inc., Madison, Wis.), witheach of the concentrations run in triplicates in a 24 well plate.Simultaneously, separate wells in the same plate will be treated withformulated BoNT/B dissolved in 1 ml of complete culture media to a finalconcentration of approximately 0.0055 nM. Cells in three replicate wellswill be treated with the contents of each resuspended formulated BoNT/C1vial. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/C1 and each formulated BoNT/C1 sample willbe calculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). Theexperimentally-derived concentration of formulated BoNT/C1 for each vialwill be extrapolated from the BoNT/C1 concentration curve using thevalue calculated from an average of three replicate wells.

3b. Assay of BoNT/C1 Activity in a Food Sample

To conduct a BoNT/C1 activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a BoNT/C1 Syntaxin-GFP substratewill be prepared, for example, using one of the methods as describedabove in Examples IV, 3 and V, 3. A standard curve will be obtained bytreating cells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM ofBoNT/C1 (Metabiologics, Inc., Madison, Wis.), with each of theconcentrations run in triplicates in a 24 well plate. Simultaneously,separate wells in the same plate will be treated with a processed foodsample from vials of formulated BoNT/C1 diluted in 1 ml of completeculture media. Cells in three replicate wells will be treated with thecontents of each diluted sample. After six hours, cells will be analyzedusing the Typhoon 9140 Imager with excitation at 484 nm and emission at510 nm. The emissions at each concentration of BoNT/C1 and eachprocessed food sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally derived concentration of BoNT/C1 present inthe processed food sample will be extrapolated from the BoNT/C1concentration curve using the value calculated from an average of threereplicate wells.

4. BoNT/B Activity Assays

4a. Assay of BoNT/D Activity in a Formulated BoNT/D Product

To conduct a BoNT/D activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/D product, cellsexpressing a BoNT/D VAMP-GFP substrate will be prepared, for example,using one of the methods as described above in Examples IV, 4 and V, 4.A standard curve will be obtained by treating cells with 0.001 nM, 0.002nM, 0.005 nM, 0.01 nM, 0.02 nM or 0.05 nM of BoNT/D (Metabiologics,Inc., Madison, Wis.), with each of the concentrations run in triplicatesin a 24 well plate. Simultaneously, separate wells in the same platewill be treated with formulated BoNT/D dissolved in 1 ml of completeculture media to a final concentration of approximately 0.0055 nM. Cellsin three replicate wells will be treated with the contents of eachresuspended formulated BoNT/D vial. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of BoNT/D andeach formulated BoNT/D sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally-derived concentration of formulated BoNT/Dfor each vial will be extrapolated from the BoNT/D concentration curveusing the value calculated from an average of three replicate wells.

4b. Assay of BoNT/D Activity in a Food Sample

To conduct a BoNT/D activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a BoNT/D VAMP-GFP substrate willbe prepared, for example, using one of the methods as described above inExamples IV, 4 and V, 4. A standard curve will be obtained by treatingcells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM of BoNT/D(Metabiologics, Inc., Madison, Wis.), with each of the concentrationsrun in triplicates in a 24 well plate. Simultaneously, separate wells inthe same plate will be treated with a processed food sample from vialsof formulated BoNT/D diluted in 1 ml of complete culture media. Cells inthree replicate wells will be treated with the contents of each dilutedsample. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/D and each processed food sample will becalculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). The experimentallyderived concentration of BoNT/D present in the processed food samplewill be extrapolated from the BoNT/D concentration curve using the valuecalculated from an average of three replicate wells.

5. BoNT/E Activity Assays

5a. Assay of BoNT/E Activity in a Formulated BoNT/E Product

To conduct a BoNT/E activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/E product,differentiated SK-N-DZ cells expressing a BoNT/E SNAP25₂₀₆-GFP substratewill be prepared, for example, using one of the methods as describedabove in Examples IV, 5 and V, 5. A standard curve will be obtained bytreating SK-N-DZ cells with 0.001 nM, 0.002 nM, 0.005 nM, 0.01 nM, 0.02nM or 0.05 nM of BoNT/E (Metabiologics, Inc., Madison, Wis.), with eachof the concentrations run in triplicates in a 24 well plate.Simultaneously, separate wells in the same plate will be treated withformulated BoNT/E dissolved in 1 ml of complete DMEM media to a finalconcentration of approximately 0.0055 nM. Cells in three replicate wellswill be treated with the contents of each resuspended formulated BoNT/Evial. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/E and each formulated BoNT/E sample willbe calculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). Theexperimentally-derived concentration of formulated BoNT/E for each vialwill be extrapolated from the BoNT/E concentration curve using the valuecalculated from an average of three replicate wells.

5b. Assay of BoNT/E Activity in a Food Sample

To conduct a BoNT/E activity assay using a food sample such as, e.g., aprocessed food sample, differentiated SK-N-DZ cells expressing a BoNT/ESNAP25₂₀₆-GFP substrate will be prepared, for example, using one of themethods as described above in Examples IV, 5 and V, 5. A standard curvewill be obtained by treating SK-N-DZ cells with 0.001, 0.002, 0.005,0.01, 0.02 or 0.05 nM of BoNT/E (Metabiologics, Inc., Madison, Wis.),with each of the concentrations run in triplicates in a 24 well plate.Simultaneously, separate wells in the same plate will be treated with aprocessed food sample from vials of formulated BoNT/E diluted in 1 ml ofcomplete DMEM media. Cells in three replicate wells will be treated withthe contents of each diluted sample. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of BoNT/E andeach processed food sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally derived concentration of BoNT/E present inthe processed food sample will be extrapolated from the BoNT/Econcentration curve using the value calculated from an average of threereplicate wells.

6. BoNT/F Activity Assays

6a. Assay of BoNT/F Activity in a Formulated BoNT/F Product

To conduct a BoNT/F activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/F product, cellsexpressing a BoNT/F VAMP-GFP substrate will be prepared, for example,using one of the methods as described above in Examples IV, 6 and V, 6.A standard curve will be obtained by treating cells with 0.001 nM, 0.002nM, 0.005 nM, 0.01 nM, 0.02 nM or 0.05 nM of BoNT/F (Metabiologics,Inc., Madison, Wis.), with each of the concentrations run in triplicatesin a 24 well plate. Simultaneously, separate wells in the same platewill be treated with formulated BoNT/F dissolved in 1 ml of completeculture media to a final concentration of approximately 0.0055 nM. Cellsin three replicate wells will be treated with the contents of eachresuspended formulated BoNT/F vial. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of BoNT/F andeach formulated BoNT/F sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally-derived concentration of formulated BoNT/Ffor each vial will be extrapolated from the BoNT/F concentration curveusing the value calculated from an average of three replicate wells.

6b. Assay of BoNT/F Activity in a Food Sample

To conduct a BoNT/F activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a BoNT/F VAMP-GFP substrate willbe prepared, for example, using one of the methods as described above inExamples IV, 6 and V, 6. A standard curve will be obtained by treatingcells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM of BoNT/F(Metabiologics, Inc., Madison, Wis.), with each of the concentrationsrun in triplicates in a 24 well plate. Simultaneously, separate wells inthe same plate will be treated with a processed food sample from vialsof formulated BoNT/F diluted in 1 ml of complete culture media. Cells inthree replicate wells will be treated with the contents of each dilutedsample. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/F and each processed food sample will becalculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). The experimentallyderived concentration of BoNT/F present in the processed food samplewill be extrapolated from the BoNT/F concentration curve using the valuecalculated from an average of three replicate wells.

7. BoNT/G Activity Assays

7a. Assay of BoNT/G Activity in a Formulated BoNT/G Product

To conduct a BoNT/G activity assay using a formulated botulinumneurotoxin product such as, e.g., a formulated BoNT/G product, cellsexpressing a BoNT/G VAMP-GFP substrate will be prepared, for example,using one of the methods as described above in Examples IV, 7 and V, 7.A standard curve will be obtained by treating cells with 0.001 nM, 0.002nM, 0.005 nM, 0.01 nM, 0.02 nM or 0.05 nM of BoNT/B (Metabiologics,Inc., Madison, Wis.), with each of the concentrations run in triplicatesin a 24 well plate. Simultaneously, separate wells in the same platewill be treated with formulated BoNT/G dissolved in 1 ml of completeculture media to a final concentration of approximately 0.0055 nM. Cellsin three replicate wells will be treated with the contents of eachresuspended formulated BoNT/G vial. After six hours, cells will beanalyzed using the Typhoon 9140 Imager with excitation at 484 nm andemission at 510 nm. The emissions at each concentration of BoNT/G andeach formulated BoNT/G sample will be calculated as a percentage of theuntreated control (fluorescence measured at 510 nm of non-toxin treatedcells). The experimentally-derived concentration of formulated BoNT/Gfor each vial will be extrapolated from the BoNT/G concentration curveusing the value calculated from an average of three replicate wells.

7b. Assay of BoNT/G Activity in a Food Sample

To conduct a BoNT/G activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a BoNT/G VAMP-GFP substrate willbe prepared, for example, using one of the methods as described above inExamples IV, 7 and V, 7. A standard curve will be obtained by treatingcells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM of BoNT/G(Metabiologics, Inc., Madison, Wis.), with each of the concentrationsrun in triplicates in a 24 well plate. Simultaneously, separate wells inthe same plate will be treated with a processed food sample from vialsof formulated BoNT/G diluted in 1 ml of complete culture media. Cells inthree replicate wells will be treated with the contents of each dilutedsample. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of BoNT/G and each processed food sample will becalculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). The experimentallyderived concentration of BoNT/G present in the processed food samplewill be extrapolated from the BoNT/G concentration curve using the valuecalculated from an average of three replicate wells.

8. TeNT Activity Assays

8a. Assay of TeNT Activity in a Formulated TeNT Product

To conduct a TeNT activity assay using a formulated botulinum neurotoxinproduct such as, e.g., a formulated TeNT product, cells expressing aTeNT VAMP-GFP substrate will be prepared, for example, using one of themethods as described above in Examples IV, 8 and V, 8. A standard curvewill be obtained by treating cells with 0.001 nM, 0.002 nM, 0.005 nM,0.01 nM, 0.02 nM or 0.05 nM of TeNT (Metabiologics, Inc., Madison,Wis.), with each of the concentrations run in triplicates in a 24 wellplate. Simultaneously, separate wells in the same plate will be treatedwith formulated TeNT dissolved in 1 ml of complete culture media to afinal concentration of approximately 0.0055 nM. Cells in three replicatewells will be treated with the contents of each resuspended formulatedTeNT vial. After six hours, cells will be analyzed using the Typhoon9140 Imager with excitation at 484 nm and emission at 510 nm. Theemissions at each concentration of TeNT and each formulated TeNT samplewill be calculated as a percentage of the untreated control(fluorescence measured at 510 nm of non-toxin treated cells). Theexperimentally-derived concentration of formulated TeNT for each vialwill be extrapolated from the TeNT concentration curve using the valuecalculated from an average of three replicate wells.

8b. Assay of TeNT Activity in a Food Sample

To conduct a TeNT activity assay using a food sample such as, e.g., aprocessed food sample, cells expressing a TeNT VAMP-GFP substrate willbe prepared, for example, using one of the methods as described above inExamples IV, 8 and V, 8. A standard curve will be obtained by treatingcells with 0.001, 0.002, 0.005, 0.01, 0.02 or 0.05 nM of TeNT(Metabiologics, Inc., Madison, Wis.), with each of the concentrationsrun in triplicates in a 24 well plate. Simultaneously, separate wells inthe same plate will be treated with a processed food sample from vialsof formulated TeNT diluted in 1 ml of complete culture media. Cells inthree replicate wells will be treated with the contents of each dilutedsample. After six hours, cells will be analyzed using the Typhoon 9140Imager with excitation at 484 nm and emission at 510 nm. The emissionsat each concentration of TeNT and each processed food sample will becalculated as a percentage of the untreated control (fluorescencemeasured at 510 nm of non-toxin treated cells). The experimentallyderived concentration of TeNT present in the processed food sample willbe extrapolated from the TeNT concentration curve using the valuecalculated from an average of three replicate wells.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A cell population comprising cells wherein greater than 50% of thecells comprise engineered cells containing: a) an exogenous Clostridialtoxin substrate comprising i) a single fluorescent member; ii) amembrane targeting domain; and iii) a Clostridial toxin recognitionsequence comprising a Clostridial toxin recognition cleavage site,wherein the cleavage site intervenes between the fluorescent member andthe membrane targeting domain; and b) a receptor that binds aClostridial toxin; wherein the engineered cells are capable ofClostridial toxin intoxication.
 2. The cell population of claim 1,wherein the engineered cells transiently contains the exogenousClostridial toxin substrate.
 3. The cell population of claim 1, whereinthe engineered cells stably contains the exogenous Clostridial toxinsubstrate.
 4. The cell population of claim 1, wherein the Clostridialtoxin substrate is expressed from a nucleic acid molecule.
 5. The cellpopulation of claim 1, wherein the cells comprising the cell populationare neuronal cells.
 6. The cell population of claim 1, wherein the cellscomprising the cell population are non-neuronal cells.
 7. The cellpopulation of claim 1, wherein the fluorescent member is a fluorescentprotein or a fluorophore binding protein.
 8. The cell populationaccording to claim 1, wherein the membrane targeting domain comprises aninterhelical loop region of synaptosomal-associated protein of 25kDa(SNAP-25), an amino-terminal α-helix region of SNAP-25, or acarboxy-terminal α-helix region of SNAP-25.
 9. The cell populationaccording to claim 1, wherein the Clostridial toxin recognition sequencecomprises a BoNT/A recognition sequence including a BoNT/A cleavagesite, a BoNT/B recognition sequence including a BoNT/B cleavage site, aBoNT/C1 recognition sequence including a BoNT/C1 cleavage site, a BoNT/Drecognition sequence including a BoNT/D cleavage site, a BoNT/Erecognition sequence including a BoNT/E cleavage site, a BoNT/Frecognition sequence including a BoNT/F cleavage site, a BoNT/Grecognition sequence including a BoNT/G cleavage site, or a TeNTrecognition sequence including a TeNT cleavage site.
 10. The cellpopulation according to claim 1, wherein the Clostridial toxinrecognition sequence comprises at least six consecutive residues ofSNAP-25, said six consecutive residues comprising Gln-Arg.
 11. The cellpopulation according to claim 1, wherein the Clostridial toxinrecognition sequence comprises at least six consecutive residues ofvesicle-associated membrane protein (VAMP), said six consecutiveresidues comprising GIn-Phe.
 12. The cell population according to claim1, wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of SNAP-25, said six consecutive residuescomprising Arg-Ala.
 13. The cell population according to claim 1,wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of Syntaxin, said six consecutive residuescomprising Lys-Ala.
 14. The cell population according to claim 1,wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of VAMP, said six consecutive residuescomprising Lys-Leu.
 15. The cell population according to claim 1,wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of SNAP-25, said six consecutive residuescomprising Arg-Ile.
 16. The cell population according to claim 1,wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of VAMP, said six consecutive residuescomprising Gln-Lys.
 17. The cell population according to claim 1,wherein the Clostridial toxin recognition sequence comprises at leastsix consecutive residues of VAMP, said six consecutive residuescomprising Ala-Ala.
 18. The cell population of claim 1, wherein thereceptor is an endogenous Clostridial toxin receptor.
 19. The cellpopulation of claim 1, wherein the receptor is an exogenous Clostridialtoxin receptor.